WO2022111644A1

WO2022111644A1 - Salt and crystal form of nitrogen-containing heterocyclic derivative, preparation method therefor and application thereof

Info

Publication number: WO2022111644A1
Application number: PCT/CN2021/133653
Authority: WO
Inventors: 刘庆鑫; 呙临松
Original assignee: 上海翰森生物医药科技有限公司; 江苏豪森药业集团有限公司
Priority date: 2020-11-26
Filing date: 2021-11-26
Publication date: 2022-06-02
Also published as: AU2021386339A1; EP4253376A1; CN116490188A; JP2023551006A; CA3200164A1; TW202229278A

Abstract

A salt and a crystal form relating to a nitrogen-containing heterocyclic derivative, a preparation method therefor and an application thereof. In particular, the present invention relates to a salt and crystal form of a compound represented by general formula (I), a preparation method therefor, a pharmaceutical composition comprising a therapeutically effective amount of the crystal form, and a use thereof as a KRAS G12C mutation inhibitor in the treatment of diseases or conditions such as leukemia, neuroblastoma, melanoma, breast cancer, lung cancer and colon cancer. Each substituent in the general formula (I) is the same as defined in the description.

Description

Salts, crystal forms of nitrogen-containing heterocyclic derivatives and their preparation methods and applications

This application claims the priority of Chinese patent application 2020113542899 with the filing date of 2020/11/26 and Chinese patent application 2021113892168 with the filing date of 2021/11/22. This application cites the full text of the above Chinese patent application.

technical field

The invention belongs to the field of biomedicine, and in particular relates to a salt, a crystal form of a nitrogen-containing heterocyclic derivative and a preparation method and application thereof.

Background technique

Rat sarcoma (rat sarcoma, RAS), encoded by the proto-oncogenes HRAS, NRAS and KRAS, is divided into four proteins HRAS, NRAS, KRAS4A and KRAS4B, which is a GTP (guanosine triphosphate) binding protein. RAS is located on the inner surface of the cell membrane, and the upstream is receptor tyrosine kinase (RTK). After activation, it regulates the downstream signaling pathways such as PI3K and RAF, thereby regulating cell growth, survival, migration and differentiation functions.

There are two main states of RAS in the body: an inactive state bound to GDP (guanosine diphosphate) and an activated state bound to GTP. Its activity is regulated by two proteins: guanine nucleotide exchange factor (GEF) promotes the release of GDP from RAS protein, enabling GTP to bind to activate RAS; GTPase activating protein (GAP) activates GTP of RAS protein Enzymatic activity, hydrolyzes GTP bound to RAS protein into GDP, and inactivates RAS. Under normal circumstances, the RAS protein is in an inactive state. After mutation, the conformation changes, RAS is in a continuously activated state, and the downstream signaling pathways are also continuously activated, which leads to the occurrence of various cancers.

As the first oncogene to be identified, RAS is the oncogene with the highest mutation rate, accounting for an average of 25% of human cancers. The most common oncogenic mutation in the RAS family was KRAS (85%), while NRAS (12%) and HRAS (3%) were less common. KRAS mutations are mainly high in a series of cancers including pancreatic cancer (95%), colorectal cancer (52%) and lung cancer (31%). The most common mutation of KRAS is point mutation, which mostly occurs in G12, G13 and Q61 of Switch II region (aa59-76) in p-loop (aa 10-17), among which G12 mutation is the most common (83%). KRAS G12C is one of the most common mutations in non-small cell lung cancer (NSCLC) and colorectal cancer.

Although there is a great clinical demand, there is no drug directly targeting KRAS on the market so far. Currently, patients with KRAS mutation can generally only be treated with chemotherapy. The research and development of KRAS inhibitors is mainly due to two factors. First, the structure of RAS protein is smooth, and it is difficult for small molecules to bind to the protein surface. Second, the affinity of RAS GTPase for GTP is as high as picomolar (pM) level, and the level of endogenous GTP is high. Small molecule drugs are difficult to block the combination of the two. Recent studies have found that KRAS 12-position glycine (Glycine, Gly) is mutated to cysteine (Cysteine, Cys), the conformation changes, forming a new pocket for covalent binding of small molecules, irreversibly locking KRAS G12C in the GDP-binding protein. inactive state. Therefore, KRAS G12C inhibitors are expected to be the first drugs to directly target KRAS.

At present, several KRAS G12C inhibitors have entered the clinical research stage, such as AMG 510 developed by Amgen, ARS-3248 developed by Wellspring Biosciences and MTRX849 developed by Mirati. A KRAS G12C inhibitor developed and marketed.

There is currently no specific targeted drug for KRAS G12C, and there is a large clinical demand. KRAS G12C inhibitors with higher selectivity, better activity and better safety have the potential to treat a variety of cancers and have broad market prospects.

The patent application (application number: PCT/CN2020/093285) of Jiangsu Hansoh Pharmaceutical Group Co., Ltd. discloses the structure of a series of pyridazine derivative inhibitors. Dry, easy to store, long-term product stability, high bioavailability, the present invention has carried out comprehensive research on the salts and crystal forms of the above substances, and is committed to obtaining the most suitable crystal form.

SUMMARY OF THE INVENTION

All the contents of the patent application PCT/CN2020/093285 are incorporated into the present application by way of reference.

The object of the present invention is to provide a kind of acid salt of compound shown in general formula (I):

in:

R ^a is each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb , haloalkoxy or hydroxyalkyl;

R ₁ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb , haloalkoxy or hydroxyalkyl;

R ₂ is selected from alkyl;

R ₃ is selected from hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb or hydroxyalkyl;

R ₄ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb or hydroxyalkyl;

R ₅ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , - NR _aa R _bb or hydroxyalkyl;

R ₆ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , - NR _aa R _bb or hydroxyalkyl;

R ₇ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, alkyl, deuterated alkyl, haloalkyl, alkoxy, -SR _aa , -C(O)R _aa , - NR _aa R _bb or hydroxyalkyl;

R _{aa is} selected from deuterium, halogen, alkyl, deuterated alkyl or haloalkyl;

R _bb is selected from deuterium, halogen, alkyl, deuterated alkyl or haloalkyl; and

x is selected from 0, 1, 2 or 3.

In a preferred embodiment of the present invention, in the acid salt of the compound represented by the general formula (I), R ^a is independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C _{1 -6} alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb , C _{1- 6} haloalkoxy or C _1-6 hydroxyalkyl;

Preferred are hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, _nitro , C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb , C _1-3 haloalkoxy or C _1-3 hydroxyalkyl;

More preferably hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated Isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, ethylthio, propylthio, isopropylthio, halomethoxy, haloethoxy, halopropoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

R ₁ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkoxy , -SR _aa , -C(O)R _aa , -NR _aa R _bb , C _1-6 haloalkoxy or C _1-6 hydroxyalkyl;

Preferred are hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb , C _1-3 haloalkoxy or C _1-3 hydroxyalkyl;

More preferably hydrogen, methyl, fluorine, chlorine, amino, hydroxyl or cyano;

R ₂ is selected from hydrogen or C _1-6 alkyl;

Preferably hydrogen or C _1-3 alkyl;

More preferably hydrogen, methyl, ethyl, propyl or isopropyl;

R ₃ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkoxy , -SR _aa , -C(O)R _aa , -NR _aa R _bb or C _1-6 hydroxyalkyl;

Preferred are hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, -SR _aa or C _1-3 hydroxyalkyl;

More preferably hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl radical, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, -S(CH ₃ ), hydroxymethyl, hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

R ₄ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkoxy , -SR _aa , -C(O)R _aa , -NR _aa R _bb , or C _1-6 hydroxyalkyl;

Preferred are hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, -NR _aa R _bb , or C _1-3 hydroxyalkyl;

More preferably hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterated propyl radical, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, -NH(CH ₃ ), -N(CH ₃ ) ₂ , hydroxymethyl, hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

R ₅ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkane oxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb or C _1-6 hydroxyalkyl;

Preferably hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, _nitro , C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy or C _1-3 hydroxyalkyl;

More preferably hydrogen, deuterium, fluorine, chlorine, bromine, iodine, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, propyl, isopropyl, deuterated methyl, deuterated ethyl, deuterium propyl, deuterated isopropyl, halomethyl, haloethyl, halopropyl, haloisopropyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxymethyl hydroxyethyl, hydroxypropyl or hydroxyisopropyl;

R ₆ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkane oxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb or C _1-6 hydroxyalkyl;

R ₇ is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkane oxy, -SR _aa , -C(O)R _aa , -NR _aa R _bb or C _1-6 hydroxyalkyl;

R _{aa is} selected from deuterium, halogen, C _1-6 alkyl, C _1-6 deuterated alkyl or C _1-6 haloalkyl;

Preferably deuterium, halogen, C _1-3 alkyl, C _1-3 deuterated alkyl or C _1-3 haloalkyl;

More preferably methyl, ethyl, propyl or isopropyl;

R _{bb is} selected from deuterium, halogen, C _1-6 alkyl, C _1-6 deuterated alkyl or C _1-6 haloalkyl;

More preferably methyl, ethyl, propyl or isopropyl;

x is selected from 0, 1, 2 or 3; preferably 0, 1 or 2; more preferably 0 or 1.

In a preferred embodiment of the present invention, the acid salt of the compound, the compound is shown in the general formula (II):

in:

^Ra is selected from hydrogen or methyl;

R ₁ is selected from hydrogen, fluorine, chlorine, bromine or methyl;

R ₃ is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, methyl, -S(CH ₃ ) or trifluoromethyl;

R ₄ is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, -N(CH ₃ ) ₂ , -NH(CH ₃ ) or fluorine;

R ₅ is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl;

R ₆ is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl;

_R7 is selected from hydrogen, fluorine, chlorine, bromine or methyl.

In a preferred embodiment of the present invention, the acid salt of the compound, the compound is further represented by the general formula (II-A) or (II-B):

In a preferred embodiment of the present invention, the acid salt of the compound, wherein the compound is selected from:

In a more preferred embodiment of the present invention, the acid salt of the compound, wherein the compound is selected from:

The acid in the acid salt is selected from isethionic acid, sulfuric acid, 1,5-naphthalenedisulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleic acid, adipic acid, hydrochloric acid , citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid or fumaric acid, preferably isethionic acid or sulfuric acid.

In a further preferred solution of the present invention, the number of acids is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.

In a further preferred solution of the present invention, the acid salt is a hydrate or anhydrous, and when the acid salt is a hydrate, the number of water is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.

In the most preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3- Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H) - acid salts of ketones, wherein the acid in the acid salt is selected from isethionic acid, sulfuric acid, 1,5-naphthalenedisulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, horse Acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid or fumaric acid, wherein the acid salt structure of the compound is as follows:

In a preferred solution of the present invention, the acid salt is a crystalline form; the preferred compound is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7- (6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d ] pyrimidin-2(1H)-one acid salt crystal form;

P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-iso Acid salt form of propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-iso Acid salt form of propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1 -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one acid salt crystalline form;

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1 -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one acid salt crystalline form;

More preferably isethionate crystal form, sulfate crystal form, 1,5-naphthalene disulfonate crystal form, mesylate crystal form, hydrobromide salt crystal form, phosphate crystal form, benzenesulfonic acid Salt crystal form, oxalate crystal form, maleate crystal form, adipate crystal form, hydrochloride crystal form, citrate crystal form, malonate crystal form, L-malate crystal form , Palmoate crystal form, p-toluenesulfonate crystal form or fumarate crystal form.

In a preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro) is provided -2-Fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)- The crystalline form of the acid salt of a ketone.

In a more preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro -2-Fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)- The acid salt of ketone is a crystal form, preferably isethionate crystal form, sulfate crystal form, 1,5-naphthalene disulfonate crystal form, mesylate crystal form, hydrobromide salt crystal form, Phosphate crystal form, benzene sulfonate crystal form, oxalate crystal form, maleate crystal form, adipate crystal form, hydrochloride crystal form, citrate crystal form, malonate crystal form , L-malate crystal form, palmoate crystal form, p-toluenesulfonate crystal form or fumarate crystal form.

In a preferred embodiment of the present invention, the acid salt is a crystalline form, wherein the number of acids is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.

In a preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro) is provided -2-Fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H)- Isethionate Forms I-III and Sulfate Forms I-IV of Ketones:

The X-ray powder diffraction pattern 2θ of isethionate Form I has a diffraction peak at 21.7±0.2°; or a diffraction peak at 8.8±0.2°; or a diffraction peak at 19.3±0.2°; or Has a diffraction peak at 27.6±0.2°; or has a diffraction peak at 10.9±0.2°; or has a diffraction peak at 15.4±0.2°; or has a diffraction peak at 16.7±0.2°; or has a diffraction peak at 15.8±0.2° or has a diffraction peak at 17.5±0.2°; or has a diffraction peak at 23.8±0.2°; or has a diffraction peak at 10.2±0.2°; or has a diffraction peak at 11.8±0.2°; preferably comprises Any 2-5 places, or 3-5 places, or 3-6 places, or 3-8 places, or 5-8 places, or 6-8 places among the above-mentioned diffraction peaks, more preferably including any 6 places, 7 places among them or 8;

The X-ray powder diffraction pattern 2θ of isethionate crystal form II has a diffraction peak at 21.7±0.2°; or has a diffraction peak at 8.8±0.2°; or has a diffraction peak at 19.3±0.2°; or Has a diffraction peak at 27.6±0.2°; or has a diffraction peak at 10.9±0.2°; or has a diffraction peak at 23.8±0.2°; or has a diffraction peak at 16.7±0.2°; or has a diffraction peak at 15.4±0.2° Have a diffraction peak; or have a diffraction peak at 15.8±0.2°; or have a diffraction peak at 10.0±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks , or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

The X-ray powder diffraction pattern 2θ of isethionate crystal form III has a diffraction peak at 19.4±0.2°; or has a diffraction peak at 16.9±0.2°; or has a diffraction peak at 26.6±0.2°; or Has a diffraction peak at 14.6±0.2°; or has a diffraction peak at 28.0±0.2°; or has a diffraction peak at 25.6±0.2°; or has a diffraction peak at 20.7±0.2°; or has a diffraction peak at 12.8±0.2° Have a diffraction peak; or have a diffraction peak at 19.1±0.2°; or have a diffraction peak at 27.2±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks , or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

The X-ray powder diffraction pattern 2θ of sulfate crystal form I has a diffraction peak at 19.0±0.2°; or a diffraction peak at 19.4±0.2°; or a diffraction peak at 12.4±0.2°; or a diffraction peak at 26.2±0.2 or a diffraction peak at 17.6±0.2°; or a diffraction peak at 18.1±0.2°; or a diffraction peak at 25.3±0.2°; or a diffraction peak at 8.8±0.2°; Or have a diffraction peak at 21.9±0.2°; or have a diffraction peak at 11.5±0.2°; preferably include any 2-5, or 3-5, or 3-6, or 3- 8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

The X-ray powder diffraction pattern 2θ of sulfate crystal form II has a diffraction peak at 15.5±0.2°; or a diffraction peak at 11.1±0.2°; or a diffraction peak at 8.9±0.2°; or a diffraction peak at 19.3±0.2 or a diffraction peak at 22.3±0.2°; or a diffraction peak at 23.6±0.2°; or a diffraction peak at 17.4±0.2°; or a diffraction peak at 27.3±0.2°; Or have a diffraction peak at 17.0±0.2°; or have a diffraction peak at 27.9±0.2°; preferably include any 2-5, or 3-5, or 3-6, or 3- 8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

The X-ray powder diffraction pattern 2θ of sulfate crystal form III has a diffraction peak at 19.6±0.2°; or has a diffraction peak at 18.0±0.2°; or has a diffraction peak at 18.4±0.2°; or has a diffraction peak at 16.8±0.2 or a diffraction peak at 14.3±0.2°; or a diffraction peak at 11.8±0.2°; or a diffraction peak at 14.9±0.2°; or a diffraction peak at 25.7±0.2°; Or have a diffraction peak at 15.4±0.2°; or have a diffraction peak at 23.5±0.2°; preferably include any 2-5, or 3-5, or 3-6, or 3- 8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

The X-ray powder diffraction pattern 2θ of sulfate crystal form IV has a diffraction peak at 19.4±0.2°; or has a diffraction peak at 18.9±0.2°; or has a diffraction peak at 15.5±0.2°; or has a diffraction peak at 8.8±0.2 or a diffraction peak at 18.1±0.2°; or a diffraction peak at 24.9±0.2°; or a diffraction peak at 17.4±0.2°; or a diffraction peak at 12.3±0.2°; Or have a diffraction peak at 26.1±0.2°; or have a diffraction peak at 14.5±0.2°; preferably include any 2-5, or 3-5, or 3-6, or 3- 8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them.

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3- Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H) - Isethionate Forms I-III and Sulfate Forms I-IV of Ketones:

The X-ray powder diffraction pattern of isethionate crystal form I at least contains one or more diffraction peaks located at 2θ of 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, preferably two of them , more preferably three strips; optionally, it can further include 2θ at 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 10.2±0.2°, 11.8±0.2° At least one of °, preferably including 2, 3, 4 or 5 of them; for example,

21.7±0.2°, 8.8±0.2°;

8.8±0.2°, 27.6±0.2°;

21.7±0.2°, 8.8±0.2°, 10.9±0.2°;

8.8±0.2°, 19.3±0.2°, 15.4±0.2°;

21.7±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

8.8±0.2°, 19.3±0.2°, 15.4±0.2°, 16.7±0.2°;

15.8±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

11.7±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

16.7±0.2°, 8.8±0.2°, 19.3±0.2°, 16.7±0.2°, 10.9±0.2°, 15.4±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 15.8±0.2°, 10.9±0.2°, 15.4±0.2°;

10.9±0.2°, 8.8±0.2°, 10.2±0.2°, 27.6±0.2°, 10.9±0.2°, 15.8±0.2°;

The X-ray powder diffraction pattern of isethionate crystal form II contains at least one or more diffraction peaks located at 2θ of 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, preferably two of them , more preferably including three; optionally, it may further include at least one located at 2θ of 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, preferably including 2 bar, 3, 4 or 5; for example,

21.7±0.2°, 10.0±0.2°;

10.0±0.2°, 8.8±0.2°;

21.7±0.2°, 10.0±0.2°, 19.3±0.2°;

10.0±0.2°, 8.8±0.2°, 27.6±0.2°;

21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°;

10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°;

27.6±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 16.7±0.2°, 10.9±0.2°;

21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 16.7±0.2°, 27.6±0.2°, 10.9±0.2°;

The X-ray powder diffraction pattern of isethionate crystal form III contains at least one or more diffraction peaks located at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, preferably two of them , more preferably including three; optionally, it can further include at least one located at 2θ of 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, preferably including 2 bar, 3, 4 or 5; for example,

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 12.8±0.2°, 28.0±0.2°, 25.6±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form I at least comprises one or more diffraction peaks located at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, preferably two of them, more preferably comprising Three; optionally, it may further comprise at least one of 2θ of 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, preferably two or three of them , 4 or 5; for example,

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 25.3±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form II at least contains one or more diffraction peaks located at 2θ of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, preferably two of them, more preferably Three; optionally, it can further comprise at least one of 2θ of 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, preferably two or three of them , 4 or 5; for example,

15.5±0.2°, 11.1±0.2°;

11.1±0.2°, 8.9±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°;

11.1±0.2°, 8.9±0.2°, 19.3±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 27.3±0.2°;

8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form III contains at least one or more diffraction peaks located at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, preferably two of them, more preferably three; optionally, it can further comprise at least one of 2θ of 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, preferably two or three of them , 4 or 5; for example,

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 14.9±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form IV contains at least one or more diffraction peaks located at 2θ of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, preferably two of them, more preferably Three; optionally, it may further comprise at least one of 2θ of 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, preferably two or three of them , 4 or 5; for example,

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 12.3±0.2°, 24.9±0.2°.

The X-ray powder diffraction pattern of isethionate Form I optionally further comprises positions at 2θ of 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 19.3±0.2° One or more diffraction peaks in 0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°; preferably at least any of 2-3, or 4-5, or 6 -8 places; further preferably, including any 2, 3, 4, 5, 6, 7 or 8 places; for example,

8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.8±0.2°, 17.5±0.2°;

27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 17.5±0.2°, 15.8±0.2°, 16.7±0.2°, 17.5±0.2°, 23.8±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 17.5±0.2°, 16.7±0.2°, 15.8±0.2°;

The X-ray powder diffraction pattern of isethionate Form II optionally further comprises positions at 2θ of 10.0±0.2°, 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9+ One or more diffraction peaks in ±0.2°, 23.8±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; more preferably, any 2, 3 at, 4, 5, 6, 7, or 8; for example,

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9+±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°;

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9+±0.2°, 23.8±0.2°, 15.4±0.2°, 15.8±0.2°, 10.0±0.2°;

The X-ray powder diffraction pattern of isethionate Form III optionally further comprises positions at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2° One or more diffraction peaks in 0.2°, 20.7±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; more preferably, any 2 or 3 of them are included , 4, 5, 6, 7 or 8; for example,

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 27.2±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 27.2±0.2°, 20.7±0.2°, 12.8±0.2°;

The X-ray powder diffraction pattern of sulfate Form I optionally further comprises positions at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3 One or more diffraction peaks in ±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; more preferably, any 2, 3, 4, 5, 6, 7 or 8; for example,

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°;

The X-ray powder diffraction pattern of the sulfate crystal form II optionally further comprises positions at 2θ of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4 One or more diffraction peaks in ±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; more preferably, any 2, 3, 4, 5, 6, 7 or 8; for example,

15.5±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°;

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form III optionally further comprises positions at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9 One or more diffraction peaks in ±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; more preferably, any 2, 3, 4, 5, 6, 7 or 8; for example,

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 15.4±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 23.5±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form IV optionally further comprises positions at 2θ of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4 One or more diffraction peaks in ±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; more preferably, any 2, 3, 4, 5, 6, 7 or 8; for example,

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 26.1±0.2°;

The X-ray powder diffraction pattern of isethionate crystalline form I comprises positions at 2θ of 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.1±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 13.3±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 17.5±0.2°, 23.8±0.2°, 14.7±0.2°, 24.3±0.2°, One or more diffraction peaks in 27.3±0.2°, 23.4±0.2°, 20.6±0.2°, 21.2±0.2°, preferably, including optional 4, 5, 6, 8 or 10 There are diffraction peaks at; for example,

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 20.6±0.2°;

19.3±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°;

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 20.6±0.2°;

19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 16.7±0.2°, 23.4±0.2°, 20.6±0.2°;

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 16.7±0.2°, 15.8±0.2°, 17.5±0.2°, 24.3±0.2°, 14.7±0.2°, 27.3±0.2°, 23.4±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 24.3±0.2°, 23.8±0.2°;

8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.1±0.2°, 27.6±0.2°, 10.9±0.2°, 13.3±0.2°, 21.2±0.2°, 15.8±0.2°, 17.5±0.2°;

The X-ray powder diffraction pattern of isethionate crystalline form II comprises positions at 2θ of 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, One or more of 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 17.5±0.2°, 14.7±0.2°, 24.4±0.2°, 27.3±0.2°, 29.2±0.2° Diffraction peaks, preferably, including optional 4, 5, 6, 8 or 10 diffraction peaks; for example,

10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 29.2±0.2°;

8.8±0.2°, 27.6±0.2°, 27.3±0.2°, 29.2±0.2°;

21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 29.2±0.2°;

10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 27.3±0.2°, 14.7±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 27.3±0.2°, 17.5±0.2°;

19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 15.8±0.2°, 16.7±0.2°, 15.4±0.2°, 17.5±0.2°;

10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 17.5±0.2°;

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 17.5±0.2°, 27.3±0.2°, 29.2±0.2°;

The X-ray powder diffraction pattern of isethionate crystalline form III comprises positions at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, One or more of 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°, 24.4±0.2°, 15.3±0.2°, 26.2±0.2°, 30.2±0.2°, 27.4±0.2° Diffraction peaks, preferably, including optional 4, 5, 6, 8 or 10 diffraction peaks; for example,

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 28.0±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 27.4±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 30.2±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 27.4±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 30.2±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 24.4±0.2°;

The X-ray powder diffraction pattern of sulfate crystalline form I contains positions at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2° One or more diffraction peaks in , 8.8±0.2°, 21.9±0.2°, 11.5±0.2°, preferably, including optional 4, 5, 6, 8 or 10 diffraction peaks ;E.g,

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 17.6±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 11.5±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 21.9±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 21.9±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form II contains 2θ at 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2° , 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 15.8±0.2°, 24.2±0.2°, 21.8±0.2°, 10.3±0.2°, 20.6±0.2° one or more diffraction peaks, Preferably, there are diffraction peaks at optional 4, 5, 6, 8 or 10 positions; for example,

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 21.8±0.2°;

15.5±0.2°, 8.9±0.2°, 22.3±0.2°, 10.3±0.2°;

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 20.6±0.2°, 27.9±0.2°;

15.5±0.2°, 11.1±0.2°, 19.3±0.2°, 22.3±0.2°, 21.8±0.2°, 10.3±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 20.6±0.2°, 27.9±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 23.6±0.2°, 17.4±0.2°, 10.3±0.2°, 20.6±0.2°;

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 20.6±0.2°;

15.5±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 15.8±0.2°, 10.3±0.2°;

The X-ray powder diffraction pattern of the sulfate crystal form III comprises at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2° , 25.7±0.2°, 15.4±0.2°, 23.5±0.2°, 18.8±0.2°, 24.7±0.2°, 9.5±0.2°, 8.8±0.2°, 11.1±0.2° one or more diffraction peaks, Preferably, there are diffraction peaks at optional 4, 5, 6, 8 or 10 positions; for example,

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 14.3±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.1±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 8.8±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 11.1±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 8.8±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 23.5±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 18.8±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form IV contains 2θ at 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2° , 12.3±0.2°, 26.1±0.2°, 14.5±0.2°, 22.2±0.2°, 24.3±0.2°, 21.7±0.2°, 23.6±0.2° one or more diffraction peaks, preferably, including wherein Diffraction peaks at optional 4, 5, 6, 8, or 10; for example,

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 18.1±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 23.6±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 21.7±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 23.6±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 14.5±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 24.3±0.2°.

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3 -Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H )-ketone isethionate crystal form I, using Cu-Kα radiation, X-ray characteristic diffraction peaks expressed in 2θ angle and interplanar spacing d value are shown in Table 1.

Table 1

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The isethionate crystal form I of (1H)-ketone, its X-ray powder diffraction pattern is basically shown in Figure 1; its DSC pattern is basically shown in Figure 2; its TGA pattern is basically shown in Figure 3.

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7 shown in Example 13-1 -(6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] The isethionate crystal form II of pyrimidin-2(1H)-one, using Cu-Kα radiation, the characteristic X-ray diffraction peaks expressed by the 2θ angle and the interplanar spacing d are shown in Table 2.

Table 2

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The isethionate crystal form II of (1H)-ketone, its X-ray powder diffraction pattern is basically shown in Figure 4; its DSC pattern is basically shown in Figure 5; its TGA pattern is basically shown in Figure 6.

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7 shown in Example 13-1 -(6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] Isethionate crystal form III of pyrimidin-2(1H)-one, using Cu-Kα radiation, the characteristic X-ray diffraction peaks represented by 2θ angle and interplanar spacing d value are shown in Table 3.

table 3

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The isethionate crystal form III of (1H)-ketone, its X-ray powder diffraction pattern is basically shown in Figure 7; its DSC pattern is basically shown in Figure 8; its TGA pattern is basically shown in Figure 9.

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7 shown in Example 13-1 -(6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] The sulfate crystal form I of pyrimidin-2(1H)-one, using Cu-Kα radiation, the characteristic X-ray diffraction peaks represented by the 2θ angle and the interplanar spacing d are shown in Table 4.

Table 4

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The sulfate crystal form I of (1H)-ketone, its X-ray powder diffraction pattern is basically as shown in FIG. 10 .

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7 shown in Example 13-1 -(6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] The sulfate crystal form II of pyrimidin-2(1H)-one, using Cu-Kα radiation, the characteristic X-ray diffraction peaks represented by the 2θ angle and the interplanar spacing d are shown in Table 5.

table 5

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The sulfate crystal form II of (1H)-ketone, and its X-ray powder diffraction pattern is basically as shown in FIG. 11 .

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7 shown in Example 13-1 -(6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] The sulfate crystal form III of pyrimidin-2(1H)-one, using Cu-Kα radiation, the characteristic X-ray diffraction peaks represented by the 2θ angle and the interplanar spacing d are shown in Table 6.

Table 6

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The sulfate crystal form III of (1H)-ketone, and its X-ray powder diffraction pattern is basically as shown in FIG. 12 .

In a further preferred embodiment of the present invention, the compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7 shown in Example 13-1 -(6-Amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] The sulfate crystal form IV of pyrimidin-2(1H)-one, using Cu-Kα radiation, the characteristic X-ray diffraction peaks represented by the 2θ angle and the interplanar spacing d value are shown in Table 7.

Table 7

The compound shown in Example 13-1 of the present invention P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino -3-Chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 The sulfate crystal form IV of (1H)-ketone, its X-ray powder diffraction pattern is basically as shown in FIG. 13 .

In a further preferred solution of the present invention, the 2θ error between the diffraction peak positions of the top ten diffraction peaks with the relative peak intensity in the X-ray powder diffraction pattern of isethionate crystal form I and the diffraction peaks at the corresponding positions in FIG. 1 is ±0.2° ~±0.5°, preferably ±0.2°～±0.3°, most preferably ±0.2°;

In the X-ray powder diffraction pattern of isethionate crystal form II, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 4 are ±0.2°～±0.5°, preferably ±0.2 °～±0.3°, most preferably ±0.2°;

In the X-ray powder diffraction pattern of isethionate crystal form III, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 7 are ±0.2°～±0.5°, preferably ±0.2 °～±0.3°, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form I, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 10 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form II, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 11 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form III, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 12 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form IV, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 13 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In a further preferred solution of the present invention, the acid salt of the compound is characterized in that the crystal form of the acid salt is a hydrate or an anhydrate, and when the crystal form of the acid salt is a hydrate, the number of water is 0.2-3 , preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3; further, the water in the hydrate is pipeline water or crystal water or a combination of both.

In a further preferred version of the present invention, the method for preparing an acid salt comprises the steps:

1) Weigh an appropriate amount of free base, add solvent to dissolve;

2) Add an appropriate amount of acid and stir; the amount of acid is preferably 1.2 equivalents;

3) quick centrifugation or standing to obtain the salt of the compound;

The solvent is an organic solvent, preferably ethanol, 2-methyltetrahydrofuran, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane at least one of;

Acid selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid, dichloro Acetic acid, trichloroacetic acid, acetylhydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetylaminobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, mandelic acid, pyrovalley Amino acid, tartaric acid, dodecyl sulfate, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid , 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethanesulfonic acid, Benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid.

In a further preferred version of the present invention, the preparation method of the acid salt of the compound and its crystal form comprises the following steps:

1) Weigh an appropriate amount of free base, add reaction solvent to dissolve;

3) after centrifugal drying, obtain the crystal form of compound acid salt;

The reaction solvent is an organic solvent, preferably ethanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, At least one of ethyl acetate or 1,4-dioxane;

In a further preferred version of the present invention, the preparation method of the acid salt crystal form of the compound comprises the following steps:

1) Weigh an appropriate amount of the salt of the compound, add an organic solvent to suspend;

2) stirring, and after centrifugal drying, the crystal form of the acid salt of the compound is obtained;

The organic solvent is selected from ethanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or at least one of 1,4-dioxane.

In a further preferred scheme of the present invention, the preparation method of the acid salt of the compound or its crystal form comprises the following steps:

1) Weigh an appropriate amount of free base, add reaction solvent to dissolve;

2) Add an appropriate amount of acid and organic solvent and stir to dissolve;

3) optionally, adding seed crystals;

4) Cool, filter the precipitated solid, wash with solvent, and dry.

The reaction solvent used in step 1) is an organic solvent, preferably ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, At least one of n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;

The acid in step 2) is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethane Sulfonic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetylaminobenzoic acid, 4-aminobenzoic acid, capric acid, Caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, Mandelic acid, pyroglutamic acid, tartaric acid, dodecyl sulfate, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentian acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methyl alcohol Sulfonic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-amino water succinic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably hydrochloric acid, phosphoric acid, Ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid.

The organic solvent in step 2) is selected from one or more of alcohols, ethers, ketones or ester solvents, preferably ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl alcohol at least one of tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;

The solvent in step 3) is selected from one or more of alcohols, ethers, ketones or ester solvents, preferably ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl alcohol At least one of tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane.

The present invention also provides a preferred solution, and also relates to a pharmaceutical composition, which comprises a therapeutically effective dose of the acid salt of the compound of general formula (I) or its crystal form, and one or more pharmaceutical acceptable carrier, diluent or excipient.

The present invention further relates to the application of any of the acid salts of the compounds of general formula (I) or their crystalline forms or the pharmaceutical compositions in the preparation of KRAS inhibitor medicines; preferably in the preparation of KRAS G12C mutation inhibitor medicines Applications.

In some embodiments, the pharmaceutically acceptable salts of the compounds of the present invention, and crystalline forms or compositions thereof, are used in the treatment of Noonan syndrome, Leopard skin syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer , head and neck cancer, gastric cancer, lung cancer and its application in colon cancer; preferably non-small cell lung cancer, colon cancer, esophageal cancer, head and neck cancer.

Detailed description of the invention

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms alkyl, most preferably an alkyl group of 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethyl pentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2 ,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl Ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethyl Alkylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof, etc. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-Methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylpropyl butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl base, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group, the present invention is preferably methyl, ethyl, isopropyl, tert-butyl, haloalkyl , deuterated alkyl, alkoxy substituted alkyl and hydroxy substituted alkyl.

The term "alkylene" means that one hydrogen atom of the alkyl group is further substituted, for example: "methylene" means _-CH2- , "ethylene" means -( _CH2 ) _2- , "propylene" Refers to -(CH ₂ ) ₃ -, "butylene" refers to -(CH ₂ ) ₄ - and the like. The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3 -Butenyl, etc. Alkenyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups, preferably cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl and cycloheptyl.

The term "spirocycloalkyl" refers to a 5- to 20-membered monocyclic polycyclic group sharing one carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings are fully conjugated π electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, spirocycloalkyl groups are classified into mono-spirocycloalkyl groups, double-spirocycloalkyl groups or poly-spirocycloalkyl groups, preferably mono-spirocycloalkyl groups and double-spirocycloalkyl groups. More preferably, it is 3-membered/6-membered, 3-membered/5-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospirocycloalkyl. Non-limiting examples of spirocycloalkyl include:

Wait;

Also included are spirocycloalkyl groups in which a monospirocycloalkyl group shares a spiro atom with a heterocycloalkyl group, non-limiting examples include:

Wait.

The term "fused cycloalkyl" refers to an all-carbon polycyclic group of 5 to 20 members in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may contain one or more rings. Multiple double bonds, but none of the rings have a fully conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicycloalkyl. Non-limiting examples of fused cycloalkyl groups include:

Wait.

The term "bridged cycloalkyl" refers to an all-carbon polycyclic group of 5 to 20 members, any two rings sharing two non-directly connected carbon atoms, which may contain one or more double bonds, but none of the rings have complete Conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl include:

The cycloalkyl ring can be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring linked to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthalene base, benzocycloheptyl, etc. Cycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2) heteroatoms, excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably contains 3 to 8 ring atoms; most preferably contains 3 to 8 ring atoms; further preferably 3-8 contains 1-3 nitrogen atoms A membered heterocyclic group, optionally substituted with 1-2 oxygen atoms, sulfur atoms, oxo groups, includes a nitrogen-containing monocyclic heterocyclic group, a nitrogen-containing spiro heterocyclic group or a nitrogen-containing fused heterocyclic group.

Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine group, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, azetyl, 1,4-diazepanyl, pyranyl, etc., preferably pyrrolidinyl, morpholinyl, piperidinyl, azetyl, 1,4-diazepanyl and piperazinyl. Polycyclic heterocyclic groups include spiro, condensed and bridged heterocyclic groups; the spiro, condensed and bridged heterocyclic groups are optionally connected with other groups through a single bond, or through a ring Any two or more atoms above are further cyclo-linked to other cycloalkyl, heterocyclyl, aryl and heteroaryl groups.

The term "spiroheterocyclyl" refers to a 5- to 20-membered monocyclic polycyclic heterocyclic group sharing one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O ) _m (where m is an integer from 0 to 2) heteroatoms and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, spiroheterocyclyls are classified into mono-spiroheterocyclyl, bis-spiroheterocyclyl or poly-spiroheterocyclyl, preferably mono-spiroheterocyclyl and bis-spiroheterocyclyl. More preferably 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl. Non-limiting examples of spiroheterocyclyl include:

Wait.

The term "fused heterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more of the rings may contain one or more Double bonds, but none of the rings have a fully conjugated pi-electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), the remaining rings Atom is carbon. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclyl groups include:

Wait.

The term "bridged heterocyclyl" refers to a 5- to 14-membered, polycyclic heterocyclyl group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings have a complete common A pi-electron system of a yoke in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl groups include:

Wait.

The heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

Wait.

Heterocyclyl may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, preferably 6 to 12 membered, such as benzene base and naphthyl. More preferred is phenyl. The aryl ring can be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, including benzo 5-10 membered heteroaryl, benzo 3-8 membered cycloalkyl and benzo 3-8 membered Heteroalkyl, preferably benzo 5-6 membered heteroaryl, benzo 3-6 membered cycloalkyl and benzo 3-6 membered heteroalkyl, wherein the heterocyclic group contains 1-3 nitrogen atoms, oxygen atoms, A heterocyclic group of sulfur atom; or a three-membered nitrogen-containing fused ring containing a benzene ring.

Wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

Wait.

Aryl may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio, carboxyl or carboxylate.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 12-membered, more preferably 5- or 6-membered, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, triazolyl, tetrazolyl , pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably triazolyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl, pyrrole base and oxazolyl. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring linked to the parent structure is a heteroaryl ring, non-limiting examples of which include:

Wait.

Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above, preferably alkyl groups containing 1 to 8 carbon atoms, more preferably An alkyl group of 1 to 6 carbon atoms, most preferably an alkyl group of 1 to 3 carbon atoms. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.

The term "alkylthio" refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Preferred are alkyl groups containing 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. Non-limiting examples of alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio. Alkylthio can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.

"Alkylthio-alkyl" means an alkylthio group attached to an alkyl group, wherein alkyl and alkylthio are as defined above.

"Alkylaminocarbonyl" means (alkyl)-N-C(O)-, wherein alkyl is as defined above.

"Haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

"Haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

"Haloalkoxy" refers to an alkylthio group substituted with one or more halogens, wherein alkylthio is as defined above.

"Hydroxyalkyl" refers to an alkyl group substituted with hydroxy, wherein alkyl is as defined above.

"Alkenyl" refers to an alkenyl group, also known as an alkenyl group, preferably an alkyl group containing 2 to 8 carbon atoms, more preferably an alkyl group of 2 to 6 carbon atoms, most preferably an alkyl group of 2 to 3 carbon atoms . The alkenyl group may be further substituted by other related groups, such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

"Alkynyl" refers to (CH≡C-), preferably an alkyl group containing 2 to 8 carbon atoms, more preferably an alkyl group of 2 to 6 carbon atoms, and most preferably an alkyl group of 2 to 3 carbon atoms. The alkynyl group may be further substituted by other related groups, such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "alkenylcarbonyl" refers to -C(O)-(alkenyl), wherein alkenyl is as defined above. Non-limiting examples of alkenylcarbonyl include: vinylcarbonyl, propenylcarbonyl, butenylcarbonyl. Alkenylcarbonyl can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.

"Hydroxy" refers to the -OH group.

"Halogen" refers to fluorine, chlorine, bromine or iodine.

"Amino" refers to _-NH2 .

"Cyano" refers to -CN.

"Nitro" refers to _-NO2 .

"Carbonyl" refers to -C(O)-.

"Carboxyl" refers to -C(O)OH.

"THF" refers to tetrahydrofuran.

"EtOAc" refers to ethyl acetate.

"MeOH" refers to methanol.

"DMF" refers to N,N-dimethylformamide.

"DIPEA" refers to diisopropylethylamine.

"TFA" refers to trifluoroacetic acid.

"MeCN" means acetonitrile.

"DMA" refers to N,N-dimethylacetamide.

" _Et2O " refers to diethyl ether.

"DCE" refers to 1,2 dichloroethane.

"DIPEA" refers to N,N-diisopropylethylamine.

"NBS" refers to N-bromosuccinimide.

"NIS" refers to N-iodosuccinimide.

"Cbz-Cl" refers to benzyl chloroformate.

"Pd2(dba ₎₃ _" refers to tris(dibenzylideneacetone)dipalladium.

"Dppf" refers to 1,1'-bisdiphenylphosphinoferrocene.

"HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate.

"KHMDS" refers to potassium hexamethyldisilazide.

"LiHMDS" refers to lithium bistrimethylsilylamide.

"MeLi" refers to methyl lithium.

"n-BuLi" refers to n-butyllithium.

"NaBH(OAc) ₃ " refers to sodium triacetoxyborohydride.

"X is selected from A, B, or C", "X is selected from A, B and C", "X is A, B or C", "X is A, B and C" etc. all express the same Meaning, that means X can be any one or more of A, B, and C.

The hydrogen atom in the present invention can be replaced by its isotope deuterium, and any hydrogen atom in the example compounds involved in the present invention can also be replaced by deuterium atom.

"Optional" or "optionally" means that the subsequently described event or circumstance can, but need not, occur, and that the description includes instances where the event or circumstance occurs or instances where it does not. For example, "heterocyclic group optionally substituted with alkyl" means that an alkyl group may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group .

"Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of one another, are substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the person skilled in the art can determine (either experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (eg, olefinic) bonds.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, and other components such as a physiological/pharmaceutically acceptable carrier and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of active ingredients and then exert biological activity.

"Pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, which are safe and effective when used in mammals, and have the desired biological activity.

Description of drawings

Figure 1 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid XRPD representation of salt Form I.

Figure 2 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid DSC representation of salt Form I.

Figure 3 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid Schematic representation of the TGA of salt Form I.

Figure 4 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid XRPD representation of salt form II.

Figure 5 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid DSC diagram of salt Form II.

Figure 6 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid TGA representation of salt form II.

Figure 7 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid XRPD representation of salt form III.

Figure 8 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid DSC diagram of salt Form III.

Figure 9 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one isethionsulfonic acid Schematic representation of the TGA of salt form III.

Figure 10 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one sulfate crystal form I Graph of XRPD.

Figure 11 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one sulfate crystal form II Graph of XRPD.

Figure 12 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one sulfate crystal form III Graph of XRPD.

Figure 13 is P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl )-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one sulfate crystal form IV Graph of XRPD.

Detailed ways

The present invention is further described below in conjunction with the examples, but these examples do not limit the scope of the present invention.

1. Preparation of Compounds

Example

The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts ([delta]) are given in parts per million (ppm). NMR was measured by Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated methanol (CD ₃ OD) and deuterated chloroform (CDCl ₃ ), and the internal standard was four Methylsilane (TMS).

An Agilent 1200 Infinity Series mass spectrometer was used for LC-MS measurements. The HPLC measurement was performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini _C18 150×4.6mm column).

The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the specifications used for TLC are 0.15mm~0.20mm, and the specifications used for TLC separation and purification products are 0.4mm~0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

The starting materials in the examples of the present invention are known and commercially available, or can be synthesized using or according to methods known in the art.

Unless otherwise specified, all the reactions of the present invention are carried out under continuous magnetic stirring, in a dry nitrogen or argon atmosphere, the solvent is a dry solvent, and the reaction temperature is in degrees Celsius.

Example 1

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl) -4-(Methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

The first step: the preparation of 4-chloro-2-(prop-1-en-2-yl)pyridin-3-amine

2,4-Dichloropyridin-3-amine (4.5 g, 27.78 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3, 2-dioxaborolane (5.13 g, 30.56 mmol), potassium carbonate (11.5 g, 83.34 mmol), Pd(PPh ₃ ) ₄ were added to dioxane (120 mL), the reaction solution was uniformly mixed, and then placed in an oil bath Stir overnight under heating at 100°C. Concentrated under reduced pressure, the obtained crude product was separated and purified by flash silica gel column chromatography to obtain the target compound 4-chloro-2-(prop-1-en-2-yl)pyridin-3-amine as a colorless oily liquid (4.5 g, yield 96%).

MS m/z(ESI): 169.1[M+H] ⁺ .

The second step: preparation of 4-(methylthio)-2-(prop-1-en-2-yl)pyridin-3-amine

4-Chloro-2-(prop-1-en-2-yl)pyridin-3-amine (2 g, 11.9 mmol), sodium methanethiolate (10 mL, 20% aqueous solution) were added to dioxane (3 mL) , the reaction solution was uniformly mixed, reacted at 100°C for 2 days, cooled to room temperature, concentrated under reduced pressure, and the obtained crude product was separated and purified by flash silica gel column chromatography to obtain compound 4-(methylthio)-2-(propan-1 -En-2-yl)pyridin-3-amine as a pale yellow liquid (1.7 g, 79% yield).

MS m/z(ESI): 181.2[M+H] ⁺ .

The third step: preparation of 2-isopropyl-4-(methylthio)pyridin-3-amine

Methanol (50 mL) was added to 4-(methylthio)-2-(prop-1-en-2-yl)pyridin-3-amine (2 g, 11.11 mmol) and Pd/C (4 g), and the reaction solution was uniformly mixed After that, it was reacted at room temperature overnight, and concentrated under reduced pressure. The obtained crude product was added to a solution mixed with methanol (5 mL), N,N-diisopropylethylamine (0.5 mL) and acrylonitrile (1 mL), and reacted at room temperature for 2 hours. Concentrated under reduced pressure, the obtained crude product was separated and purified by flash silica gel column chromatography to obtain compound 2-isopropyl-4-(methylthio)pyridin-3-amine as a colorless liquid (500 mg, yield 25%).

MS m/z(ESI): 183.2[M+H] ⁺ .

The fourth step: preparation of 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide

To 2,6-dichloro-5-fluoronicotinamide (500 mg, 2.44 mmol) and oxalyl chloride (1.32 mL, 2.54 mmol) was added THF (10 mL), the reaction solution was uniformly mixed, and then reacted at 60°C for 3 hours, The reaction temperature was lowered to room temperature, triethylamine (680 mg, 6.6 mmol), 2-isopropyl-4-(methylthio) pyridin-3-amine (400 mg, 2.2 mmol) were added, the reaction was carried out at room temperature for 1 hour, and concentrated under reduced pressure. , the obtained crude product was separated and purified by flash silica gel column chromatography to obtain the compound 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl) ) nicotinamide as a white solid (800 mg, 87% yield).

MS m/z(ESI): 417.1[M+H] ⁺ .

Step 5: 7-Chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2 Preparation of (1H)-ketones

2,6-Dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (800 mg, 1.92 mmol) was added to THF ( 20mL), after the reaction solution was uniformly mixed, KHMDS (4.8mL, 4.8mmol) was slowly added at 0°C, reacted at room temperature for 1 hour, concentrated under reduced pressure, and the obtained crude product was separated and purified by flash silica gel column chromatography to obtain compound 7-chloro -6-Fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one in white Solid (600 mg, 82% yield).

MS m/z(ESI): 381.1[M+H] ⁺ .

The sixth step: tert-butyl (S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl- Preparation of 1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate

7-Chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H) - Ketone (300mg, 0.79mmol), phosphorus oxychloride (600mg, 3.95mmil), DIPEA (1g, 7.9mmol) were added with THF (40mL), the reaction solution was mixed uniformly and reacted at 80°C for 1 hour. The reaction temperature Drop to room temperature, slowly add tert-butyl (S)-3-methylpiperazine-1-carboxylate (240 mg, 1.19 mmol) to the reaction solution, react at room temperature for 1 hour, concentrate under reduced pressure, and the obtained crude product is rapidly Silica gel column chromatography separation and purification to obtain the compound tert-butyl (S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)- 2-Carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate as a white solid (400 mg, 90% yield).

MS m/z(ESI): 563.1[M+H] ⁺ .

The seventh step: (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio) Preparation of pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

tert-Butyl(S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2 - Dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (400 mg, 0.71 mmol), TFA ( ₂ mL) was added _CH2Cl2 (30 mL) , the reaction solution was uniformly mixed, reacted at room temperature for 1 hour, concentrated under reduced pressure, CH ₂ Cl ₂ (20 mL) and DIPEA (0.3 mL) were added to the obtained crude product, the reaction temperature was lowered to 0 ° C, and slowly added to the reaction solution Acryloyl chloride (0.1 mL) was reacted at room temperature for 1 hour, and concentrated under reduced pressure. The obtained crude product was separated and purified by flash silica gel column chromatography to obtain compound (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-iso Propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was a yellow solid (200 mg, 55% yield).

MS m/z(ESI): 517.1[M+H] ⁺ .

The eighth step: 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2 Preparation of -isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine -3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (50 mg, 0.1 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (30 mg, 0.2 mmol), Pd ( dppf)Cl ₂ (16mg, 0.02mmol), cesium carbonate (100mg, 0.3mmol) was added to dixoane (1.5mL), after the reaction solution was uniformly mixed, the reaction was heated under microwave heating at 100 ° C for 1 hour, and concentrated under reduced pressure. CH ₂ Cl ₂ (20 mL) and DIPEA (0.3 mL) were added, the reaction temperature was lowered to 0° C., and acryloyl chloride (0.1 mL) was slowly added to the reaction solution, reacted at room temperature for 1 hour, and concentrated under reduced pressure. The obtained crude product was separated and purified by Pre-HPLC to obtain the compound 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl) )-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one as a white solid (14 mg, yield: twenty four%).

MS m/z(ESI): 593.1[M+H] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.40 (d, J=5.6Hz, 1H), 8.22-8.27 (m, 1H), 7.21-7.27 (m, 2H), 6.79-6.88 (m, 1H) ), 6.58-6.66(m, 2H), 6.28-6.34(m, 1H), 5.84(d, J=12.0Hz, 1H), 5.06(s, 1H), 4.43-4.59(m, 2H), 4.07- 4.23(s, 1H), 3.57-3.85(m, 2H), 3.20-3.48(m, 1H), 2.79-2.85(m, 1H), 2.41(s, 3H), 1.47(d, J=4.8Hz, 3H), 1.20(d, J=6.4Hz, 3H), 1.06(d, J=6.8Hz, 3H).

Example 1-1 and Example 1-2

(P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2- Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((S)-4-acryloyl) -2-Methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridine-3 -yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

Example 1 Two axial chiral isomers were obtained by SFC resolution Example 1-1 and Example 1-2, SFC: Chiral preparation conditions:

仪器instrument	SFC-150(Thar,Waters)SFC-150 (Thar, Waters)
柱型 Cylindrical		IC 20250mm,10μm(Daicel)IC 20250mm,10μm(Daicel)
柱压column pressure	100bar100bar
流动相mobile phase	CO ₂/Methanol(0.2％Methanol Ammonia)＝50/50 CO ₂ /Methanol(0.2%Methanol Ammonia)=50/50
流速flow rate	120g/min120g/min
检测波长Detection wavelength	UV 214nmUV 214nm
柱温column temperature	35℃35℃

Example 1-1:

t _R =1.92min

MS m/z(ESI): 593.1[M+H] ⁺ .

Example 1-2:

t _R = 2.43min

MS m/z(ESI): 593.1[M+H] ⁺ .

¹ HNMR (400MHz, MeOD-d ₄ ) δ 8.40 (d, J=5.6Hz, 1H), 8.25 (t, J=10.8Hz, 1H), 7.21-7.27 (m, 2H), 6.79-6.90 (m ,1H),6.58-6.66(m,2H),6.28-6.34(m,1H),5.83(dd,J=10.8Hz,2.0Hz,1H),5.05-5.10(m,1H),4.41-4.57( m,2H),4.07-4.21(m,1H),3.61-3.87(m,2H),3.24-3.36(m,1H),2.77-2.83(m,1H),2.41(s,3H),1.46- 1.49(m, 3H), 1.19(d, J=6.8Hz, 3H), 1.06(d, J=6.8Hz, 3H).

Example 2

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

The first step: 4,7-Dichloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2( Preparation of 1H)-ketone

7-Chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2( 1H)-ketone (200mg, 0.526mmol) in acetonitrile (10mL) solution was added N,N-diisopropylethylamine (407mg, 3.16mmol), followed by phosphorus oxychloride (242mg, 1.58mmol), stirring at 80°C 1 hour. It was cooled to room temperature and used directly in the next reaction.

The second step: tert-butyl(2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2- Preparation of carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate

N,N-diisopropylethylamine (678mg, 5.26mmol) and tert-butyl (2R,5S)-2,5-dimethylpiperazine-1-carboxylate (224mg) were added to the reaction solution in the previous step. , 1.005mmol), and stirred at room temperature for 1 hour. Add water (60 mL), extract with ethyl acetate (40 mL×3), wash the organic phase with aqueous ammonium chloride solution (40 mL), then wash with aqueous sodium chloride solution (30 mL), and concentrate after column chromatography [eluent: two Chloromethane ~ methanol/dichloromethane from 0% to 2.2%] purification to give tert-butyl (2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-( Methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxy Acid ester (200 mg, 66% yield over 2 steps) as a yellow solid.

MS m/z(ESI): 577.2[M+H] ⁺ , 579.2[M+H+2] ⁺

The third step: 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio) Preparation of pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate

To tert-butyl(2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1 , 2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (200 mg, 0.347 mmol) in dichloromethane (6 mL) Trifluoroacetic acid (1.2 mL) was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated at low temperature to obtain 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methyl) Thio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate (200 mg) as a red oil, used quickly for the next reaction.

MS m/z (ESI): 477.2[M+H] ⁺ , 479.2[M+H+2] ⁺ .

The fourth step: 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl- Preparation of 4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

To 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine -3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate (200 mg, 0.347 mmol) in dichloromethane (15 mL) was added N,N-diisopropyl ethylamine (447 mg, 3.47 mmol), acryloyl chloride (63 mg, 0.694 mmol) was added dropwise at 0° C., and the mixture was stirred for 1 hour. The reaction was quenched with ammonium chloride aqueous solution (30 mL), extracted with dichloromethane (30 mL × 3), the dichloromethane layer was washed with saturated aqueous NaCl solution (20 mL), dried over anhydrous sodium sulfate, concentrated and then subjected to column chromatography [eluent] : Dichloromethane ~ methanol/dichloromethane from 0% to 2.5%] purification to obtain 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7- Chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (130 mg, 2 steps Yield 71%) yellow solid.

MS m/z (ESI): 530.2[M+H] ⁺ , 532.2[M+H+2] ⁺ .

The fifth step: 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl) - Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methyl) Thio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (130 mg, 0.246 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (77 mg, 0.491 mmol) ), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (40 mg, 0.0491 mmol) and cesium carbonate (240 mg, 0.738 mmol) were added to dioxane ( 8 mL) and water (1 mL), replaced with nitrogen, and stirred with microwave at 100°C for 1 hour. The reaction solution was concentrated and purified by column chromatography [eluent: dichloromethane～methanol/dichloromethane from 0% to 2.5%] to obtain 4-((2S,5R)-4-acryloyl-2,5-dimethylformaldehyde ylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridine [2,3-d]pyrimidin-2(1H)-one (90 mg, 60% yield) was a yellow solid.

MS m/z (ESI): 606.2 [M+H] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.40 (d, J=8Hz, 1H), 8.29-8.18 (m, 1H), 7.30-7.18 (m, 2H), 6.93-6.73 (m, 1H) ,6.70–6.56(m,2H),6.36–6.20(m,1H),5.89–5.75(m,1H),5.15–4.98(m,1H),4.63–4.22(m,2H),4.11–3.82( m, 2H), 3.68–3.40 (m, 1H), 2.88–2.65 (m, 1H), 2.40 (d, J=4Hz, 3H), 1.53–1.43 (m, 3H), 1.36 (t, J=8Hz) ,1H),1.28(t,J=8Hz,2H),1.23–1.16(m,3H),1.10–1.01(m,3H).

Example 2-1 and Example 2-2

(P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)- 1-(2-Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((2S, 5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl- 4-(Methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

Example 2 Two axial chiral isomers were obtained by SFC resolution Example 2-1 and Example 2-2, SFC: Chiral preparation conditions:

仪器instrument	SFC-150(Thar,Waters)SFC-150 (Thar, Waters)
柱型 Cylindrical		IC 20250mm,10μm(Daicel)IC 20250mm,10μm(Daicel)
柱压column pressure	100bar100bar
流动相mobile phase	CO ₂/Methanol(0.2％Methanol Ammonia)＝60/40 CO ₂ /Methanol(0.2%Methanol Ammonia)=60/40
流速flow rate	100g/min100g/min
检测波长Detection wavelength	UV 214nmUV 214nm
柱温column temperature	35℃35℃

Example 2-1:

t _R = 1.99min

MS m/z (ESI): 606.2 [M+H] ⁺ .

Example 2-2:

t _R = 2.87min

MS m/z (ESI): 606.2 [M+H] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.40 (d, J=8Hz, 1H), 8.27-8.18 (m, 1H), 7.30-7.19 (m, 2H), 6.94-6.74 (m, 1H) ,6.70–6.56(m,2H),6.36–6.20(d,J=16Hz,1H),5.90–5.75(m,1H),5.14–4.98(m,1H),4.63–4.22(m,2H), 4.12–3.82 (m, 2H), 3.68–3.41 (m, 1H), 2.87–2.65 (m, 1H), 2.40 (d, J=4Hz, 3H), 1.53–1.42 (m, 3H), 1.36 (t , J=8Hz, 1H), 1.28 (t, J=8Hz, 2H), 1.23–1.16 (d, J=4Hz, 3H), 1.10–1.01 (d, J=4Hz, 3H).

Example 3

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl) -4-(Methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl) Reference Example 1 for the preparation of -4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one).

MS m/z(ESI): 609.1[M+H] ⁺ .

Example 4

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylthio)phenyl)-1-(2 -Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylthio)phenyl)-1-(2 -Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) Reference Example 1.

MS m/z(ESI): 622.8[M+H] ⁺ .

Example 5

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl) Preparation of -4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

The first step: preparation of N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide

4-Chloro-3-fluoroaniline (1.45 g, 0.01 mol) was dissolved in THF (150 mL), Na ₂ CO ₃ (3.18 g, 0.03 mol) was added, under nitrogen protection, cooled to 0 ° C, and trifluoroethyl was added dropwise Acid anhydride (4.2 mL, 0.03 mol) was added dropwise and stirred at room temperature for 10 hours. The reaction solution was added to water (150 mL). Ethyl acetate (100 mL) was extracted three times. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product, which was purified by column chromatography (PE/EA=5:1) to obtain the target product N-(4-chloro-3-fluorophenyl)-2,2, 2-Trifluoroacetamide (2.3 g, 95% yield).

¹ H NMR (400 MHz, MeOD-d ₄ ) δ 7.70 (dd, J=11.1, 2.0 Hz, 1H), 7.49-7.40 (m, 2H);

¹⁹ F NMR (376 MHz, MeOD-d ₄ ) δ-77.17(s);

MS m/z (ESI): 242.1 [M+H] ⁺ .

The second step: the preparation of (6-amino-3-chloro-2-fluorophenyl) boronic acid

Dissolve N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (2.3 g, 9.5 mmol) in THF (40 mL), cool to -78 °C under nitrogen protection, dropwise n-BuLi (7.9 mL, 19.0 mmol, 2.4 M) was added, the dropwise addition was completed, and the mixture was stirred at -50°C for 50 minutes. The reaction solution was cooled to -78 ° C, triisopropyl borate (2.3 g, 9.5 mmol) (4.8 mL, 20.9 mmol) was added dropwise, the dropwise addition was completed, stirred at the same temperature for 20 minutes, removed the dry ice bath, and stirred at room temperature for 2 hours . Then, the reaction solution was cooled to 0°C, diluted hydrochloric acid (19 mL, 1M) was added dropwise, the temperature was raised to 40°C, and the mixture was stirred for 1 hour. Ethyl acetate (100 mL) was extracted three times. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product, which was purified by column chromatography (PE/EA=4:1) to obtain the target product (6-amino-3-chloro-2-fluorophenyl)boronic acid (1.1 g, 56% yield).

MS m/z (ESI): 190.0 [M+H] ⁺ .

The third step: the preparation of (2-amino-6-fluorophenyl) boronic acid

(6-amino-3-chloro-2-fluorophenyl)boronic acid (100 mg, 0.53 mmol) was dissolved in MeOH (20 mL), Pd/C (20 mg) was added, hydrogen was replaced three times, and the reaction was stirred at 15 psi for 2 hours, TLC (PE/EA 1:1) detected the reaction was complete. Filtration and concentration of the filtrate gave the target product (2-amino-6-fluorophenyl)boronic acid (80 mg, yield 97%) as a yellow solid, which was directly used in the next reaction without purification.

MS m/z (ESI): 156.0 [M+H] ⁺ .

The fourth step: 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2 Preparation of -isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine -3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl)boronic acid (23.2 mg, 0.15 mmol) and cesium carbonate (48.87 mg, 0.15 mmol) were dissolved in dioxane/ _H2O (1.5 mL/0.3 mL). The nitrogen was replaced for 1 minute, and the reaction was carried out for 1 hour under the condition of microwave at 100°C. After the reaction was completed, the reaction solution was concentrated, purified by column chromatography (CH ₂ Cl ₂ /MeOH=20:1) to obtain the crude product, and then purified by preparative HPLC to obtain the yellow solid target product 4-((S)-4-acryloyl-2- Methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl) Pyrido[2,3-d]pyrimidin-2(1H)-one (7.0 mg, 24% yield).

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.46 (d, J=5.4Hz, 1H), 8.25 (dd, J=21.2, 12.0Hz, 1H), 7.27 (d, J=5.5Hz, 1H) ,7.11(dd,J＝14.7,8.2Hz,1H),6.84(d,J＝14.2Hz,1H),6.49(d,J＝8.3Hz,1H),6.41–6.27(m,2H),5.83( dd,J＝10.6,1.6Hz,1H),4.48(dd,J＝52.4,11.6Hz,2H),4.30–3.83(m,2H),3.74(d,J＝9.7Hz,2H),3.22(s ,1H),2.98–2.80(m,1H),2.43(d,J=0.7Hz,3H),1.56–1.40(m,3H),1.22(d,J=6.6Hz,3H),1.01(d, J=6.6Hz, 3H).

¹⁹ F NMR (376MHz, MeOD-d ₄ )δ-114.58--114.95(m),-114.95--115.34(m),-125.12--126.48(m).

MS m/z (ESI): 592.2 [M+H] ⁺ .

Example 6

2-(4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine-3 -yl)-2-oxy-1,2-dihydropyridine[2,3-d]pyrimidin-7-yl)-3-fluorobenzamide

2-(4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine-3 -yl)-2-oxy-1,2-dihydropyridine[2,3-d]pyrimidin-7-yl)-3-fluorobenzamide was prepared with reference to Example 1.

MS m/z (ESI): 619.7 [M+H] ⁺ .

Example 7

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-(dimethylamino)-6-fluorophenyl)-6-fluoro-1-(2 -Isopropyl-4-(methylthio)pyridin-3-yl)pyridinyl[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-(dimethylamino)-6-fluorophenyl)-6-fluoro-1-(2 -The preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyridinyl[2,3-d]pyrimidin-2(1H)-one refers to Example 1.

MS m/z (ESI): 619.7 [M+H] ⁺ .

Example 8

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylamino)phenyl)-1-(2- Refer to Example 1 for the preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyridyl[2,3-d]pyrimidin-2(1H)-one.

MS m/z (ESI): 605.7 [M+H] ⁺ .

Example 9

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2 Preparation of -isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-chloro-1-(2-isopropyl-4-(methylthio)pyridine -3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26.7 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl)boronic acid (28.4 mg, 0.15 mmol) and potassium acetate (15.0 mg, 0.15 mmol) were dissolved in dioxane/ _H2O (1.5 mL/0.3 mL). The nitrogen was replaced for 1 minute, and the reaction was carried out for 1 hour under the condition of microwave at 100°C. After the reaction was completed, the reaction solution was concentrated, purified by column chromatography (CH ₂ Cl ₂ /MeOH=20:1) to obtain the crude product, and then purified by preparative HPLC to obtain the yellow solid target product 4-((S)-4-acryloyl-2- Methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl) Pyrido[2,3-d]pyrimidin-2(1H)-one (3.7 mg, 14% yield).

MS m/z (ESI): 642.1 [M+H] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.56-8.39 (m, 2H), 7.24 (t, J=5.3Hz, 1H), 7.15 (dd, J=15.4, 6.9Hz, 1H), 6.84 ( d, J=9.9Hz, 1H), 6.53–6.46 (m, 1H), 6.32 (d, J=15.9Hz, 1H), 5.84 (d, J=12.2Hz, 1H), 4.68–4.36 (m, 3H) ), 4.10(dd, J＝45.7, 31.6Hz, 2H), 3.76(s, 1H), 2.94(s, 2H), 2.42(d, J＝6.2Hz, 3H), 1.57–1.43(m, 3H) ,1.22(d,J＝6.7Hz,3H),1.06(dd,J＝42.4,6.7Hz,3H). ¹⁹ F NMR(376MHz,MeOD)δ-117.04–-117.24(m),-117.24–-117.51 (m).

Example 9-1 and Example 9-2

(P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2- Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((S)-4-acryloyl) -2-Methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine-3 -yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

Example 9 Two axial chiral isomers were obtained by SFC resolution Example 9-1 and Example 9-2, SFC: Chiral preparation conditions:

仪器instrument	SFC-80(Thar,Waters)SFC-80 (Thar, Waters)
柱型 Cylindrical		IC 20250mm,10μm(Daicel)IC 20250mm,10μm(Daicel)
柱压column pressure	100bar100bar

流动相mobile phase	CO ₂/Methanol(0.2％Methanol Ammonia)＝45/55 CO ₂ /Methanol(0.2%Methanol Ammonia)=45/55
流速flow rate	80g/min80g/min
检测波长Detection wavelength	UV 214nmUV 214nm
柱温column temperature	35℃35℃

Example 9-1:

t _R =1.74min

MS m/z (ESI): 642.1 [M+H] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.56-8.39 (m, 2H), 7.24 (t, J=5.3Hz, 1H), 7.15 (dd, J=15.4, 6.9Hz, 1H), 6.84 ( d, J=9.9Hz, 1H), 6.53–6.46 (m, 1H), 6.32 (d, J=15.9Hz, 1H), 5.84 (d, J=12.2Hz, 1H), 4.68–4.36 (m, 3H) ), 4.10(dd, J＝45.7, 31.6Hz, 2H), 3.76(s, 1H), 2.94(s, 2H), 2.42(d, J＝6.2Hz, 3H), 1.57–1.43(m, 3H) ,1.22(d,J＝6.7Hz,3H),1.06(dd,J＝42.4,6.7Hz,3H).

¹⁹ F NMR (376MHz, MeOD-d ₄ )δ-117.04--117.24(m),-117.24--117.51(m).

Example 9-2:

t _R = 2.49min

MS m/z (ESI): 642.1 [M+H] ⁺ .

¹ H NMR (400 MHz, MeOD-d ₄ ) δ 8.56-8.39 (m, 2H), 7.27-7.10 (m, 2H), 6.84 (dd, J=28.3, 17.7 Hz, 1H), 6.50 (d, J =8.8Hz,1H),6.32(d,J=16.9Hz,1H),5.83(d,J=11.7Hz,1H),4.63-4.41(m,2H),4.23-4.02(m,1H),3.79 –3.57(m, 2H), 3.36(s, 2H), 2.99–2.86(m, 1H), 2.41(d, J=7.6Hz, 3H), 1.51(d, J=25.9Hz, 3H), 1.21( d, J=6.6Hz, 3H), 1.05 (dd, J=44.8, 6.7Hz, 3H).

Example 10

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-fluoro-1-(2 Preparation of -isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine -3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl)boronic acid (28.4 mg, 0.15 mmol) and cesium carbonate (48.8 mg, 0.15 mmol) were dissolved in dioxane/ _H2O (1.5 mL/0.3 mL). The nitrogen was replaced for 1 minute, and the reaction was carried out for 1 hour under the condition of microwave at 100°C. After the reaction was completed, the reaction solution was rotated to dryness, purified by column chromatography (CH ₂ Cl ₂ /MeOH=20:1) to obtain the crude product, and then purified by preparative HPLC to obtain the yellow solid target product 4-((S)-4-acryloyl- 2-Methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridine-3- yl)pyrido[2,3-d]pyrimidin-2(1H)-one (4.4 mg, 14% yield).

¹ H NMR (400 MHz, MeOD-d ₄ ) δ 8.47 (d, J=5.4 Hz, 1H), 8.38-8.24 (m, 1H), 7.27 (d, J=5.4 Hz, 1H), 7.17 (t, J＝8.6Hz,1H),6.85(d,J＝14.9Hz,1H),6.49(d,J＝8.9Hz,1H),6.32(d,J＝16.3Hz,1H),5.84(d,J＝ 10.5Hz, 1H), 4.57 (d, J=23.5Hz, 2H), 4.42 (s, 1H), 4.24–3.89 (m, 2H), 3.73 (dd, J=14.4, 7.9Hz, 1H), 2.92 ( s, 1H), 2.43 (s, 3H), 1.54–1.40 (m, 3H), 1.22 (d, J=6.7Hz, 3H), 1.01 (d, J=6.6Hz, 3H).

¹⁹ F NMR (376MHz, MeOD-d ₄ )δ-116.46--116.73(m),-116.87(dd,J=39.0,8.4Hz),-126.18(dd,J=24.9,15.2Hz).

MS m/z (ESI): 626.1 [M+H] ⁺ .

Example 11

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,3-difluoro-6-hydroxyphenyl)-6-fluoro-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,3-difluoro-6-hydroxyphenyl)-6-fluoro-1-(2- Reference Example 2 for the preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 611.1[M+H] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.41 (d, J=5.6Hz, 1H), 8.32-8.25 (m, 1H), 7.25 (d, J=5.6Hz, 1H), 7.20-7.13 ( m, 1H), 6.92-6.82(m, 1H), 6.62-6.58(m, 1H), 6.34-6.28(m, 1H), 5.83(d, J=10.4Hz, 1H), 5.14-5.04(m, 1H), 4.64-4.42(m, 2H), 4.25-4.07(m, 1H), 3.89-3.61(m, 3H), 2.88-2.77(m, 1H), 2.42(s, 3H), 1.52-1.46( m,3H),1.20(d,J=6.4Hz,3H),1.05(d,J=6.4Hz,3H).

Example 12

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-chloro-1-(2-isopropyl- Preparation of 4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-chloro-1-(2-isopropyl- Reference Example 2 for the preparation of 4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 611.1[M+H] ⁺ .

Example 13

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro - Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

The first step: preparation of 2,5,6-trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide

Under the protection of _N2 , 2,5,6-trichloronicotinamide (6.2 g, 27.7 mmol) was dissolved in THF (60 mL), and oxalyl chloride (15.2 mL, 31.5 mmol) (2 M/L) was added dropwise at -78 °C. Dichloromethane solution), stirred at -78°C for 10 minutes, and stirred at 60°C for 3 hours, the reaction solution was cooled to 0°C, triethylamine (18 mL, 111 mmol) was added dropwise, 2-isopropyl-4-(methylsulfide) was added dropwise yl)pyridin-3-amine (5 g, 27.7 mmol) in THF and stirred at room temperature for 2 hours. Add brine to quench, add water and ethyl acetate (3*100mL) for extraction, combine organic layers, dry over anhydrous sodium sulfate, filter, and concentrate to obtain crude product, which is purified by column chromatography (DCM/MeOH=100:1 to 70:1) The target product 2,5,6-trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (8.6g, yield 72%) was obtained .

MS m/z(ESI): 433.1[M+H] ⁺ , 435.1[M+H+2] ⁺ .

Step 2: 6,7-Dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H, Preparation of 3H)-dione

2,5,6-Trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (10.4 g, 24.1 mmol) was dissolved in dry water In THF (80 mL), under nitrogen protection, cooled to 0°C, KHMDS (48 mL, 48.2 mmol) was added dropwise, and the mixture was stirred for 0.5 h. Quenched with saturated aqueous ammonium chloride solution, extracted with water and ethyl acetate (3*100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified by slurrying with ethyl acetate to obtain the target product 6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridine- 3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (8 g, 84% yield).

MS m/z(ESI): 397.1[M+H] ⁺ , 399.1[M+H+2] ⁺ .

Step 3: 4,6,7-Trichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2(1H) - Preparation of ketones

6,7-Dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)- The diketone (5.2 g, 13.1 mmol) was dissolved in ACN (50 mL), DIEA (23 mL, 66 mmol), POCl ₃ (3 mL, 19.7 mmol) were added, and the mixture was stirred at 80° C. for 0.5 h. used directly in the next reaction.

MS m/z(ESI): 415.1[M+H] ⁺ , 417.1[M+H+2] ⁺ .

The fourth step: tert-butyl(2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridine-3-yl)-2-carbonyl Preparation of -1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate

4,6,7-Trichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one In acetonitrile (50 mL), add DIEA (23 mL, 66 mmol), add tert-butyl (2R,5S)-2,5-dimethylpiperazine-1-carboxylate (6.2 g, 26.2 mmol), stir at room temperature 1 hour. Quenched with water, extracted with water and ethyl acetate (3*100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified by column chromatography (CH ₂ Cl ₂ /MeOH=30:1) to obtain the target product tert-butyl(2R,5S)-4-(6, 7-Dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidine-4 -yl)-2,5-dimethylpiperazine-1-carboxylate (6.1 g, 77% yield).

MS m/z(ESI): 593.1[M+H] ⁺ , 595.1[M+H+2] ⁺ .

The fifth step: 6,7-dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio) ) Preparation of pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

tert-butyl(2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1, 2-Dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (6.1 g, 10.3 mmol) was dissolved in dichloromethane (20 mL) To the solution, TFA (20 mL) was added, and the mixture was stirred at room temperature for 1 hour. Concentrate to obtain the crude target product 6,7-dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio) yl)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (6.1 g, 100% yield).

MS m/z(ESI): 493.1[M+H] ⁺ , 495.1[M+H+2] ⁺ .

The sixth step: 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4 Preparation of -(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

6,7-Dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridine- 3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (6 g, 12.2 mmol) was dissolved in dichloromethane (30 mL), DIEA (30 mL, 131 mmol) was added, acryloyl chloride (1.08 g mL, 13.13 mmol) and stirred at room temperature for 1 hour. Quenched with water, extracted with water and ethyl acetate (3*100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified by column chromatography (CH ₂ Cl ₂ /MeOH=20:1) to obtain the target product 4-((2S,5R)-4-acryloyl-2 ,5-Dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3- d] Pyrimidine-2(1H)-one (1.6 g, 22% yield).

MS m/z(ESI): 547.1[M+H] ⁺ , 549.1[M+H+2] ⁺ .

The seventh step: 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl) - Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

Under _N2 protection, 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl) -4-(Methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (700 mg, 1.3 mmol), (6-amino-3-chloro-2-fluoro Phenyl)boronic acid (380 mg, 2.6 mmol) was dissolved in a mixture of 1,4-dioxane and water (6 mL: 0.3 mL), Pd(dppf) _Cl2.DCM (100 mg, 0.1 mmol), KOAc (400mg, 4mmol), microwave reaction at 100°C for 1 hour. Quenched with water, extracted with water and ethyl acetate (3*50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified by column chromatography (CH ₂ Cl ₂ /MeOH=200:1 to 80:1) to obtain the target product 4-((2S,5R)-4- Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4 -(Methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (400 mg, 48% yield).

MS m/z(ESI): 656.1[M+H] ⁺ , 658.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.47-8.34(m,2H),7.24-7.20(m,1H),7.10-7.14(m,1H),6.79-6.68(m,1H),6.42 –6.40(d,J=8.0Hz,1H),6.24-6.17(m,1H),5.75-5.71(m,1H),5.01-4.94(m,2H),4.46-4.40(m,1H),4.26 -4.17(m,1H),4.03-3.99(m,1H),3.84-3.79(m,1H),2.86-2.77(m,1H),2.36(s,3H),1.26-1.19(m,9H) ,1.14-1.11(m,3H).

Example 13-1 and Example 13-2

(P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)- 6-Chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4- ((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1 -(2-Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

Example 13 Two axial chiral isomers were obtained by SFC resolution Example 13-1 and Example 13-2, SFC: Chiral preparation conditions:

仪器instrument	SFC-150(Thar,Waters)SFC-150 (Thar, Waters)
柱型 Cylindrical		IC 20250mm,10μm(Daicel)IC 20250mm,10μm(Daicel)
柱压column pressure	100bar100bar
流动相mobile phase	CO ₂/Methanol(0.2％Methanol Ammonia)＝40/60 CO ₂ /Methanol(0.2%Methanol Ammonia)=40/60
流速flow rate	120g/min120g/min
检测波长Detection wavelength	UV 214nmUV 214nm
柱温column temperature	35℃35℃

Example 13-1:

t _R =1.74min

MS m/z(ESI): 656.1[M+H] ⁺ , 658.1[M+H+2] ⁺ .

¹ H NMR (400MHz, MeOD-d ₄ ) δ 8.47-8.34(m, 2H), 7.24-7.20(m, 1H), 7.10-7.14(m, 1H), 6.79-6.68(m, 1H), 6.42 –6.40(d,J=8.0Hz,1H),6.24-6.17(m,1H),5.75-5.71(m,1H),5.01-4.94(m,2H),4.46-4.40(m,1H),4.26 -4.17(m,1H),4.03-3.99(m,1H),3.84-3.79(m,1H),2.86-2.77(m,1H),2.36(s,3H),1.26-1.19(m,9H) ,1.14-1.11(m,3H).

Example 13-2:

t _R = 2.49min

MS m/z(ESI): 656.1[M+H] ⁺ , 658.1[M+H+2] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.55-8.38(m, 2H), 7.25-7.20(m, 1H), 7.18-7.11(m, 1H), 6.88-6.76(m, 1H), 6.51 –6.47(d,J=8.0Hz,1H),6.33-6.27(m,1H),5.84-5.80(m,1H),5.12-5.10(m,2H),4.46-4.23(m,2H),4.15 -3.89(m,2H),3.64-3.50(m,1H),2.89-2.82(m,1H),2.43(s,3H),1.51-0.99(m,12H).

Example 14

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 623.1[M+H] ⁺ , 625.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.47-8.34(m,2H),7.21-7.20(m,2H),6.89-6.77(m,1H),6.64-6.55(m,2H),6.32 –6.26(m,1H),5.84-5.80(m,1H),5.08-5.03(m,2H),4.56-4.49(m,1H),4.34-4.26(m,1H),4.13-4.04(m, 1H), 3.92-3.88(m, 1H), 2.79-2.72(m, 1H), 2.40(s, 3H), 1.55-1.43(m, 3H), 1.35-1.27(m, 3H), 1.20-1.17( m,3H),1.08-1.05(t,J=8.0Hz,3H).

Example 14-1 and Example 14-2

(P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)- 1-(2-Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one) and (M-4-((2S, 5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl- 4-(Methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one)

Example 14 Two axial chiral isomers were obtained by SFC resolution Example 14-1 and Example 14-2, SFC: Chiral preparation conditions:

Example 14-1:

t _R = 2.46min

MS m/z(ESI): 623.1[M+H] ⁺ , 625.1[M+H+2] ⁺ .

Example 14-2:

t _R =3.08min

MS m/z(ESI): 623.1[M+H] ⁺ , 625.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.48-8.34(m,2H),7.23-7.21(m,2H),6.90-6.78(m,1H),6.66-6.58(m,2H),6.33 –6.28(m,1H),5.85-5.82(m,1H),5.10-5.06(m,2H),4.58-4.50(m,1H),4.34-4.27(m,1H),4.13-4.06(m, 1H), 3.93-3.88(m, 1H), 2.79-2.71(m, 1H), 2.41(s, 3H), 1.56-1.46(m, 3H), 1.37-1.29(m, 3H), 1.21-1.18( m,3H),1.07-1.05(t,J=8.0Hz,3H).

Example 15

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-fluoro-1-(2-isopropyl- Preparation of 4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-fluoro-1-(2-isopropyl- Reference Example 2 for the preparation of 4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 595.1[M+H] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.40-8.32(m,2H),7.51(t,J=7.6Hz,1H),7.22(d,J=5.4Hz,1H),7.05(t, J=8.4Hz, 2H), 6.86-6.79(m, 1H), 6.37-6.26(m, 1H), 5.84(d, J=10.6Hz, 1H), 5.08(m, 2H), 4.56-4.46(m ,2H),4.21-4.08(m,1H),3.85-3.62(m,2H),2.86-2.82(m,1H),2.40(s,3H),1.47(d,J=6.6Hz,3H), 1.21–1.19(d,J=6.8Hz,3H),1.04(d,J=6.8Hz,3H).

Example 16

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluorophenyl)-1-(2-isopropyl-4- Preparation of (methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluorophenyl)-1-(2-isopropyl-4- Reference Example 2 for the preparation of (methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 576.7[M+H] ⁺

Example 17

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)- Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)- Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 690.1[M+H] ⁺ ,692.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.46-8.34(m,2H),7.25-7.21(m,1H),7.11-7.14(m,1H),6.44-6.42(d,J=8.0Hz ,1H),6.23-6.16(m,1H),5.73-5.70(m,1H),5.03-4.97(m,2H),4.47-4.42(m,1H),4.25-4.16(m,1H),4.06 -4.02(m,1H),3.86-3.83(m,1H),2.84-2.79(m,1H),2.34(s,3H),1.27-1.19(m,9H),1.16-1.14(m,3H) .

Example 18

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1 Preparation of -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1 Preparation of -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 660.1[M+H] ⁺ ,662.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.58-8.38(m,2H),7.53-7.36(m,1H),7.23-7.15(m,1H),6.97-6.79(m,1H),6.22 (d, J=16Hz, 1H), 5.77 (d, J=8Hz, 1H), 5.45–5.40 (m, 2H), 5.07–4.82 (m, 1H), 4.50–3.98 (m, 3H), 3.92– 3.49 (m, 2H), 3.17–3.02 (m, 1H), 2.93–2.63 (m, 1H), 2.44–2.26 (m, 3H), 1.43–1.27 (m, 3H), 1.08 (d, J=4Hz ,3H),1.04–0.86(m,3H).

Example 19

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro- Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro- Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 640.1[M+H] ⁺ , 642.1[M+H+2] ⁺ .

¹ H NMR(400MHz, Methanol-d ₄ )δ8.57-8.35(m,3H),7.25-7.04(m,2H),6.96-6.79(m,1H),6.29-6.14(m,1H),5.77 (d, J=12Hz, 1H), 5.09–4.82 (m, 1H), 4.76–4.58 (m, 2H), 4.48–3.98 (m, 3H), 3.94–3.59 (m, 2H), 2.93–2.69 ( m, 1H), 2.44–2.29 (m, 3H), 2.10–1.95 (m, 3H), 1.42–1.26 (m, 3H), 1.08 (d, J=4Hz, 3H), 1.05–0.87 (m, 3H) ).

Example 20

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)- Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)- Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 674.1[M+H] ⁺ ,676.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.61-8.39(m,2H),7.56-7.35(m,1H),7.27-7.14(m,1H),6.96-6.75(m,1H),6.20 (d, J=16Hz, 1H), 5.82–5.71 (m, 1H), 5.53–5.38 (m, 2H), 4.95–4.69 (m, 1H), 4.57–4.30 (m, 1H), 4.24–4.00 ( m, 2H), 3.98–3.79 (m, 2H), 2.95–2.60 (m, 1H), 2.44–2.25 (m, 3H), 1.40–1.13 (m, 6H), 1.10–0.87 (m, 6H).

Example 21

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro- 1-(2-Isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro- Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 674.1[M+H] ⁺ ,

Example 22

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 626.1[M+H] ⁺ , 628.1[M+H+2] ⁺ .

¹ H NMR(400MHz, Methanol-d ₄ )δ8.58-8.34(m,2H),7.26-6.99(m,2H),6.95-6.77(m,1H),6.47-6.27(m,1H),6.26 –6.13(m,1H),5.77(d,J=16Hz,1H),5.22(s,2H),5.09–4.80(m,1H),4.50–3.99(m,3H),3.95–3.53(m, 2H), 3.20–2.98 (m, 1H), 2.94–2.65 (m, 1H), 2.42–2.24 (m, 3H), 1.43–1.25 (m, 3H), 1.09 (d, J=4Hz, 3H), 1.04–0.82(m,3H).

Example 23

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-( Refer to Example 13 for the preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z (ESI): 644.1 [M+H]+.

Example 24

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6- Preparation of chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6- Preparation of chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z (ESI): 658.1 [M+H]+.

Example 25

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl) - Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl) - Preparation of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13 .

MS m/z(ESI): 654.1[M+H] ⁺ ,656.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.58-8.31(m,2H),7.25-7.03(m,2H),6.94-6.73(m,1H),6.19(d,J=16Hz,1H) ,5.81–5.69(m,1H),4.96–4.59(m,3H),4.55–4.38(m,1H),4.29–3.96(m,2H),3.93–3.72(m,2H),3.00–2.60( m, 1H), 2.45–2.25 (m, 3H), 2.07–1.94 (m, 3H), 1.43–1.13 (m, 6H), 1.12–0.82 (m, 6H).

Example 26

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6- Preparation of chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6- Preparation of chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 658.1[M+H]+,

Example 27

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 644.1[M+H]+,

Example 28

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro- Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro- Preparation of 1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 640.2[M+H]+,

Example 29

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 626.1[M+H]+,

Example 30

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl) Preparation of yl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl) Reference Example 13 for the preparation of yl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 607.1[M+H] ⁺ , 609.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.57-8.34(m,2H),7.43-7.31(m,1H),7.18(d,J=4Hz,1H),7.15-7.01(m,2H) ,6.95–6.78(m,1H),6.28–6.14(m,1H),5.77(d,J=12Hz,1H),5.07–4.86(m,1H),4.45–4.25(m,2H),4.22– 3.98 (m, 1H), 3.93–3.58 (m, 2H), 3.21–3.02 (m, 1H), 2.87–2.69 (m, 1H), 2.40–2.27 (m, 3H), 1.98–1.85 (m, 3H) ), 1.41–1.28 (m, 3H), 1.08 (d, J=8Hz, 3H), 1.02–0.79 (m, 3H).

Example 31

4-((2S,5R)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2- Preparation of isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 621.2[M+H]+,

Example 32

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl) Preparation of -4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl) Reference Example 13 for the preparation of -4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 627.1[M+H] ⁺ ,629.1[M+H+2] ⁺ .

¹ H NMR(400MHz,Methanol-d ₄ )δ8.56-8.30(m,2H),7.58-7.36(m,3H),7.19(s,1H),6.87(s,1H),6.24-6.19(d , J=20.0Hz, 1H), 5.79-5.76(d, J=12.0Hz, 1H), 4.97(s, 1H), 4.32-4.04(m, 3H), 3.80-3.49(m, 3H), 2.72( s,1H),2.35(s,3H),1.34-0.91(m,9H).

Example 33

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 641.6[M+H]+,

Example 34

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl) Preparation of -7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl) Reference Example 13 for the preparation of -7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 589.1[M+H] ⁺ ,591.1[M+H+2] ⁺ .

Example 35

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-( Preparation of methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-( Reference Example 13 for the preparation of methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI):609.6[M+H]+,

Example 36

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-( Preparation of 2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 661.1[M+H]+,

Example 37

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridine Preparation of -3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridine Reference Example 13 for the preparation of -3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 603.1[M+H] ⁺ ,605.1[M+H+2] ⁺ .

Example 38

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl) Preparation of yl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl) Reference Example 13 for the preparation of yl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

MS m/z(ESI): 623.6[M+H]+,

Example 39

4-((2S,5R)-(4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)benzene yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((2S,5R)-(4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)benzene yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 675.1[M+H]+,

Example 40

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1 Preparation of -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1 Preparation of -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one Reference Example 13.

MS m/z(ESI): 676.1[M+H] ⁺ , 678.1[M+H+2] ⁺ .

2. Evaluation of compound biological tests

The present invention is further described and explained below in conjunction with test examples, but these examples are not meant to limit the scope of the present invention.

Test Example 1. Determination of the inhibitory effect of NCI-H358/Mia PaCa-2 cell proliferation activity

1.1 The purpose of the experiment:

The inhibitory effect of the example compounds on the proliferation activity of KRAS G12C mutant cell lines NCI-H358 and Mia PaCa-2 cells was determined.

1.2. Experimental instruments and reagents:

1.2.1 Instruments:

Microplate reader (BioTek Synergy H1)

Pipette (Eppendorf & Rainin)

1.2.2 Reagents:

NCI-H358 was purchased from Nanjing Kebai Biotechnology Co., Ltd.;

Mia PaCa-2 was purchased from ATCC;

Cell Titer-Glo cells were purchased from Promega, the product number is G7573;

RPMI 1640 was purchased from Gibco, part number 22400089;

DMEM was purchased from Gibco, catalog number 11995065;

FBS was purchased from Gibco, item number 10091148;

PBS was purchased from Gibco, catalog number 10010023;

Pancreatin was purchased from Gibco, catalog number 25200056;

Cell culture plates were purchased from Corning Company, Cat. No. 3610.

1.3. Experimental method:

When NCI-H358 or Mia PaCa-2 cells are cultured to an appropriate degree of confluency, collect NCI-H358 or Mia PaCa-2 cells, use complete medium to adjust the cells to an appropriate cell concentration, and plate the cell suspension on a 96-well plate , 90 μL per well, put it into a 37°C, 5% CO ₂ incubator overnight, use DMSO and medium to prepare compound solutions of different concentrations, set a vehicle control, add the compound solution to a 96-well plate, 10 μL per well, After culturing in a 37°C, 5% CO ₂ incubator for 72 hours, add CellTiter-Glo solution, shake and mix evenly, incubate in the dark for 10 minutes, and read with a BioTek Synergy H1 microplate reader.

1.4. Experimental data processing method:

The inhibition rate was calculated using the luminescence signal value, and the concentration and inhibition rate were fitted to a nonlinear regression curve using Graphpad Prism software to obtain the IC ₅₀ value.

1.5. Experimental results:

The experimental results are shown in Table 8, the IC ₅₀ values of the compounds of the examples on the proliferation inhibitory activity of NCI-H358 and Mia PaCa-2 cells.

Table 8

Note: "NT" means not detected.

1.6. Experimental conclusion:

According to the data, the compounds of the examples of the present invention have a good proliferation inhibitory effect on NCI-H358 and Mia PaCa-2 cells.

Test Example 2. Determination of the ability of the compound of the present invention to improve the stability (melting temperature) of KRAS G12C protein

2.1. Experiment purpose:

The ability of the test compound to enhance the stability of KRAS G12C protein (the degree of protein melting temperature increase can characterize the binding ability of the compound to KRAS G12C protein).

2.2. Experimental reagents and instruments:

2.2.1 Experimental equipment:

Quantitative PCR instrument (Quantstudio6Flex) was purchased from Life Company;

Pipettes were purchased from Eppendorf or Rainin.

2.2.2 Experimental reagents:

Protein Thermal Shift ^TM Dye Kit was purchased from Thermofisher Company, item number 4461146;

KRAS G12C protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., the product number is 12259-H07E2;

HEPES, 1M Buffer Solution was purchased from Thermofisher Company, Item No. 15630080;

DTT was purchased from Sigma, the product number is 43816-50mL;

NaCl was purchased from Sinopharm Chemical Reagent Co., Ltd., the product number is 10019318.

2.3 Experimental method:

In this experiment, the thermal shift method was used to test the degree of change in the melting temperature (Tm) of KRAS G12C protein before and after compound binding to characterize the ability of the compound to improve the stability of KRAS G12C protein.

The specific experimental operations are as follows:

A solution containing 20 μM HEPES (pH 7.5), 1 mM DTT, 5X SYPRO Orange, and 150 mM NaCl was prepared as experimental buffer while adding human KRAS G12C protein at a final concentration of 5.37 μM. Divide the above reaction mixture into 8 PCR tubes, each tube is 19.5 μL, add 0.5 μL of the test compound or DMSO respectively, the total reaction system is 20 μL, the final compound concentration is 10 μM, and 2.5% DMSO is set as the vehicle control . After incubating at room temperature for one hour in the dark, put the PCR tube into the PCR machine, open the QuantStudio Software v1.3 software, and select the melt curve function to detect the melting temperature of KRAS G12C protein in different treatment groups (heated from 25°C to 95°C, 0.03 °C/s).

2.4. Experimental data processing method:

The experimental data file of the PCR instrument was imported into the thermal shift software, and the melting temperature (Tm) of each treatment group was obtained, and the Tm of the DMSO solvent control group was subtracted to obtain the change value (ΔTm) of the melting temperature.

2.5. Experimental results:

Through the above scheme, it can be concluded that in the experiment of improving the stability of KRAS G12C protein in combination with the compound of the present invention, it shows the ability to increase the melting temperature of the protein as shown in Table 9.

Table 9

实施例编号Example number	Tm(℃)DMSOTm(℃)DMSO	Tm(℃)Tm(℃)	ΔTm(℃)ΔTm(℃)
实施例1Example 1	48.648.6	60.260.2	11.611.6
实施例2Example 2	48.748.7	57.257.2	8.58.5
实施例3Example 3	50.650.6	61.561.5	10.910.9
实施例4Example 4	49.549.5	61.261.2	11.711.7
实施例5Example 5	48.648.6	64.464.4	15.815.8
实施例9Example 9	46.846.8	60.260.2	13.413.4
实施例13Example 13	47.047.0	58.058.0	11.011.0

2.6 Experimental conclusion:

The above data show that the compounds of the examples of the present invention have good binding ability to KRAS G12C protein.

Test Example 3. Inhibitory activity of the compounds of the present invention on p-ERK in Miapaca-2 cells

3.1. Experimental purpose:

The inhibitory activity of example compounds on phosphorylated ERK levels in KRAS G12C mutant cells Mia PaCa-2 was determined.

3.2. Experimental equipment:

3.2.1 Instruments:

Microplate reader (BioTek Synergy H1);

Pipette (Eppendorf & Rainin).

3.2.2 Reagents:

Phosphorylated ERK1/2 (T202-Y204) LANCE Ultra Cellular Detection Kit was purchased from PerkinElmer, the item number is TRF4000M;

The cell culture plate was purchased from Corning Company, the product number is 3610;

White opaque OptiPlate ^™ -384 plates were purchased from PerkinElmer, Catalog No. 6007290.

3.3. Experimental method:

When the Mia PaCa-2 cells were cultured to an appropriate degree of confluency, collect the Mia PaCa-2 cells, adjust the cell density to 1×10 ⁶ /mL using complete medium, and spread the cell suspension in a 96-well plate, 50 μL per well, Put them in a 37°C, 5% CO ₂ incubator overnight, use DMSO and complete medium to prepare compound solutions of different concentrations, set a vehicle control, add compound solutions to 96-well plates, 25 μL per well, and put them at 37°C , After culturing for 2 hours in a 5% _CO2 incubator, discard the supernatant from the cell culture plate, add 50 μL of lysate to each well, shake at room temperature for 30 minutes, put it in a centrifuge at 1000 rpm for 1 minute, and transfer 15 μL of the supernatant to In a 384-well plate, add 5 μL of detection mixture (Eu-labeled anti-ERK1/2 (T202-Y204) Antibody with a final detection concentration of 0.5 nM and ULight labeled anti-ERK1/2 Antibody with a final detection concentration of 5 nM) to each well. ), centrifuge at 1000 rpm for 1 minute to mix well, react overnight at room temperature, read the plate with BioTek Synergy H1, and use a time-resolved fluorescence program to detect the signal values at 620 nm and 665 nm emission wavelengths.

3.4. Experimental data processing method:

Calculate the ratio of the signal values at the emission wavelengths of 665 nm and 620 nm, use the ratio to calculate the inhibition rate, and use the Graphpad Prism software to perform nonlinear regression curve fitting to obtain the IC ₅₀ value.

3.5. Experimental results:

Table 10 _IC50 values of pERK inhibition in Mia PaCa-2 cells

3.6. Experimental conclusion:

The above data show that the compounds of the examples of the present invention have a good inhibitory effect on pERK in Mia PaCa-2 cells.

Test Example 4. Pharmacokinetic Determination in Mice

4.1. Research purposes:

Balb/c mice were used as test animals to study the pharmacokinetic behavior of the compounds administered orally in mice (plasma).

4.2. Test scheme:

4.2.1 Test drug:

Compounds of the examples of the present invention, self-made;

4.2.2 Experimental animals:

Balb/c mice, male, were purchased from Shanghai Jisijie Laboratory Animal Co., Ltd., animal production license number (SCXK (Shanghai) 2013-0006N0.311620400001794).

4.2.3 Drug preparation:

Weigh 5g of hydroxyethyl cellulose (HEC, CMC-Na, viscosity: 800-1200Cps), dissolve in 1000mL of purified water, and add 10g of Tween80. Mix well into a clear solution.

The compounds of the examples were weighed, respectively added into 4-mL glass bottles, 2.4 mL of the solution was added, and sonicated for 10 minutes to obtain a colorless and clear solution with a concentration of 1 mg/mL.

4.2.4 Administration:

Balb/c mice, male; PO respectively after overnight fasting, the dose is 10 mg/kg, and the administration volume is 10 mL/kg.

4.2.5 Sample collection:

Blood was collected before administration and at 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, and 8h after administration. The blood was placed in an EDTA-2K test tube, centrifuged at 6000 rpm at 4°C for 6 min to separate plasma, and stored at -80°C ; 4h food after administration. 4.3 Experimental results:

The final determination results obtained by LCMS/MS method are shown in Table 11

Table 11: Mouse Pharmacokinetic Parameters of Compounds

4.4 Experimental conclusion:

The above data show that the compounds of the examples of the present invention have good pharmacokinetic parameters in mice.

Test Example 5. Tumor inhibition experiment on MiaPaca 2 xenograft model

5.1 Experimental purpose:

BALB/c nude mice were used as test animals, and human pancreatic cancer cell MiaPaca 2 xenograft (CDX) model was used to conduct in vivo efficacy experiments to evaluate the anti-tumor effects of the test compounds.

5.2 Experimental instruments and reagents:

5.2.1 Instruments:

Ultra-clean workbench (BSC-1300II A2, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory);

_CO2 incubator (Thermo-311, Thermo);

Centrifuge (Centrifuge 5720R, Eppendorf);

Automatic cell counter (Countess II, Life Technologies);

Pipette (10-20 μL, Eppendorf);

Microscope (Ts 2, Nikon);

Vernier caliper (CD-6"AX, Mitutoyo, Japan);

Cell culture flask (T25/T75/T225, Corning);

Constant temperature water tank (HWS12, Shanghai Yiheng Science).

5.2.2 Reagents:

DMEM (11995-065, Gibco);

Fetal bovine serum (FBS) (10091-148, Gibco);

0.25% trypsin (25200-056, Gibco);

Penicillin-streptomycin double antibody (P/S) (SV30010, GE);

Phosphate Buffered Saline (PBS) (10010-023, Gibco);

Matrigel(356234, Corning);

Gln (25030-081, Gibco).

5.3 Experimental operation:

Take out MiaPaca 2 cells from the cell bank, add DMEM medium (containing 10% FBS, 1% Glu, 1% P/S) to culture in a CO ₂ incubator (incubator temperature is 37°C, CO ₂ concentration 5%). The cells were subcultured after the cells covered 80-90% of the bottom of the culture flask, and the cells continued to be cultured in a CO ₂ incubator after subculture. This process was repeated until the number of cells met the inoculum requirement of the in vivo efficacy, and the cells in the logarithmic growth phase were collected and counted with an automatic cell counter. A cell suspension (density 8×10 ⁷ /mL) was prepared and placed in an ice box for use.

The animals used are BALB/c nude mice, female, 6-8 weeks old, weighing about 18-22 grams. Mice were maintained in a specific pathogen-free environment and in single ventilated cages, 5 mice per cage. All cages, bedding and water were sanitized prior to use, and all animals had free access to standard certified commercial laboratory diets. Nude mice were marked with disposable ear tags for rats and mice before the experiment, and the skin of the inoculation site was disinfected with 75% medical alcohol before inoculation. Each mouse was subcutaneously inoculated with 0.1ml (containing 8*10 ⁶ cells) of MiaPaca 2 on the right back. tumor cells. Group dosing was started when the mean tumor volume reached 100-200 ^mm3 . The test compound was administered orally by oral gavage every day, and the dosage/frequency of administration (6 mg/kg QD x 3w), the drug efficacy of each group at the end of the experiment is shown in Table 5.

5.4 Data processing:

Tumor volume (mm ³ ) was measured twice a week with a vernier caliper, and the calculation formula was: V=0.5*D*d*d, where D and d were the long and short diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the mean tumor increase volume in compound-treated animals by the mean tumor increase volume in untreated animals. The calculation formula of tumor inhibition rate is: TGI (%)=1-[(Vt-V0) administration group/(Vt-V0) solvent control group]*100%. All animals were euthanized after the experiment.

5.5 Experimental results:

Table 12: Pharmacodynamic parameters of compounds in xenografted mice

5.6 Experimental conclusion:

The above data show that: after continuous oral administration for 21 days, the compounds of the examples of the present invention can significantly inhibit the growth of MiaPaca 2 nude mice xenografts under the condition of oral daily administration of 6 mg/kg.

Test Example 6. In vivo pharmacodynamic study on human lung cancer NCI-H358 cell xenograft tumor model

6.1 Experimental purpose:

The in vivo efficacy of the compounds on the human lung cancer NCI-H358 cell xenograft tumor model was evaluated.

6.2 Experimental instruments and reagents:

6.2.1 Instruments:

1) Biological safety cabinet (BSC-1300II A2, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory);

2) Ultra-clean workbench (CJ-2F, Suzhou Fengshi Laboratory Animal Equipment Co., Ltd.);

3) _CO2 incubator (Thermo-311, Thermo);

4) centrifuge (Centrifuge 5720R, Eppendorf);

5) Automatic cell counter (Countess II, Life Technologies);

6) Vernier caliper (CD-6"AX, Mitutoyo, Japan);

7) Cell culture flask (T75/T225, Corning);

8) Electronic balance (CPA2202S, Sartorius);

9) Electronic balance (BSA2202S-CW, Sartorius);

10) Electronic balance (BS124S, Sartorius).

6.2.2 Reagents:

1) RPMI-1640 medium (22400-089, Gibco);

2) DMEM medium (11995-065, Gibco);

3) Fetal bovine serum (FBS) (10099-141C, Gibco);

4) Phosphate Buffered Saline (PBS) (10010-023, Gibco);

5) Tween 80 (30189828, Sinopharm Reagent);

6) Sodium carboxymethyl cellulose (30036365, Sinopharm Reagent.)

6.3 Experimental operation and data processing:

6.3.1 Animals:

BALB/c nude mice, 6-8 weeks, female, were purchased from Shanghai Sipple-Bike Laboratory Animal Co., Ltd.

6.3.2 Cell culture and cell suspension preparation

1) Take out a strain of MiaPaca-2 cells from the cell bank, revive the cells with DMEM medium (DMEM+10% FBS), and place the revived cells in a cell culture flask (mark the cell type, date, cultured person on the bottle wall). name, etc.) in a CO ₂ incubator (incubator temperature is 37°C, CO ₂ concentration is 5%) (the method of recovering NCI-H358 cells is the same as that of MiaPaca-2 cells in Test Example 5, and the medium is changed to RPMI-1640 culture medium).

2) Passage every three to five days, and cells continue to be cultured in a CO ₂ incubator after passage. This process is repeated until the number of cells meets the in vivo efficacy requirements.

3) Collect the cultured MiaPaca-2 cells, count them with an automatic cell counter, and resuspend the cells with PBS and Matrigel (1:1 ratio) according to the counting results to prepare a cell suspension (the cell density is 5×10 ⁷ ). /mL), placed in an ice box for use (NCI-H358 cells do not need to be added with Matrigel, they can be directly resuspended in PBS, and the cell density is 1×10 ⁸ /mL).

6.3.3 Sample configuration:

1) Solvent: solvent (0.5% CMC-Na+1% Tween 80), storage condition: 4°C.

Weigh 0.5 g of CMC-Na, add a certain volume of ddH ₂ O to dissolve, then add 1.0 mL of Tween 80, stir and mix, and finally make up to 100 mL.

2) Preparation of test compound (10 mg/kg):

Weigh 8.42 mg of AMG510 compound, add 8.260 mL of solvent, and obtain a homogeneous solution by ultrasonication, vortexing, and stirring.

7.81 mg of the compound of Example 13-1 was weighed, 7.654 mL of solvent was added, and a homogeneous solution was obtained by ultrasonication, vortexing and stirring.

6.3.3 Cell seeding

1) Label nude mice with disposable ear tags for rats and mice before inoculation;

2) Mix the cell suspension evenly during inoculation, use a 1mL syringe to extract 0.1-1mL of the cell suspension, remove air bubbles, and then place the syringe on an ice pack for later use;

3) Hold the nude mouse with the left hand, use 75% alcohol to sterilize the position of the right back of the nude mouse against the right shoulder (inoculation site), and start the inoculation after 30 seconds;

4) Inoculate the experimental nude mice in sequence (each mouse is inoculated with 0.1 mL of cell suspension);

6.3.4 Tumor measurement, grouping and administration of tumor-bearing mice:

1) According to the tumor growth, the tumor was measured on the 18th day after inoculation, and the tumor size was calculated.

Calculation of tumor volume: tumor volume (mm ³ )=length (mm)×width (mm)×width (mm)/2

2) According to the body weight and tumor size of the tumor-bearing mice, the method of random grouping was used for grouping.

3) According to the grouping results, start to administer the test drug (administration method: oral administration; administration dose: 10 mg/kg; administration volume: 10 mL/kg; administration frequency: 1 time/day; administration period: 21 days ; vehicle: 0.5% CMC/1% Tween 80).

4) Tumors were measured and weighed twice a week after the test drug was started.

5) Euthanize the animals after the experiment.

6) Use software such as Excel to process data.

6.4 Data processing:

Calculation of compound tumor inhibition rate TGI (%): when the tumor does not regress, TGI (%)=[(1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)) /(The average tumor volume at the end of the solvent control group treatment - the average tumor volume at the beginning of the solvent control group treatment)] × 100%. When the tumor has regressed, TGI (%)=[1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/average tumor volume at the beginning of administration of this treatment group]× 100%.

6.5 Experimental results:

Table 13: Pharmacodynamic parameters of compounds in xenografted mice

6.6 Experimental conclusion:

The above data show that: after 15 days of continuous oral administration, the compounds of the examples of the present invention can significantly inhibit the growth of nude mice transplanted with human lung cancer NCI-H358 cells under the condition of oral administration of 10 mg/kg per day, which is significantly better than the reference data.

Test example 7, hERG potassium channel inhibitory activity test

7.1 Cell Preparation

7.1.1 CHO-hERG cells were cultured in a 175cm ² culture flask. When the cell density had grown to 60-80%, the culture medium was removed, washed once with 7mL of PBS, and then digested with 3mL of Detachin.

7.1.2 After the digestion is complete, add 7 mL of culture medium to neutralize, then centrifuge, remove the supernatant, and add 5 mL of culture medium to resuspend to ensure the cell density is 2-5×10 ⁶ /mL.

7.2 Solution Preparation

Table 14: Composition of intracellular and extracellular fluids

7.3 Electrophysiological recording process

Single-cell high-impedance sealing and whole-cell pattern formation are all done automatically by the Qpatch instrument. After acquiring the whole-cell recording pattern, the cells are clamped at -80 mV, before a 5-second +40 mV depolarizing stimulus is given. , given a pre-voltage of -50 mV for 50 ms, then repolarized to -50 mV for 5 seconds, and then returned to -80 mV. This voltage stimulus was applied every 15 seconds, and the extracellular fluid was recorded for 2 minutes, followed by recording for 5 minutes, and then the administration process was started. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2.5 minutes. Positive control compound 3 μM Cisapride. At least 3 cells were tested at each concentration (n≥3).

7.4 Compound Preparation

7.4.1 Dilute the 20 mM compound stock solution with extracellular fluid, add 5 μL of 20 mM compound stock solution to 2495 μL of extracellular fluid, dilute 500-fold to 40 μM, and then perform 3-fold serial dilution in the extracellular fluid containing 0.2% DMSO. Obtain the final concentration to be tested.

7.4.2 The highest test concentration is 40 μM, which are 40, 13.33, 4.44, 1.48, 0.49, and 0.16 μM in order of 6 concentrations.

7.4.3 The DMSO content in the final test concentration should not exceed 0.2%, and this concentration of DMSO has no effect on the hERG potassium channel.

7.5 Data Analysis

The experimental data were analyzed by XLFit software.

7.6 Quality Control

Environment: Humidity 20～50%, Temperature 22～25℃

Reagents: The experimental reagents used were purchased from Sigma, the purity was >98%

Experimental data in the report must meet the following criteria:

Whole cell sealing impedance >100MΩ

Tail current amplitude>400pA

Pharmacological parameters:

The inhibitory effect of multiple concentrations of Cisapride on hERG channel was set as a positive control.

7.7 Experimental results:

Table 15: Inhibition results of the embodiment of the present invention on hERG current at multiple concentrations

实施例编号Example number	hERG(μM)hERG(μM)
实施例2-1Example 2-1	>30>30
实施例9-1Example 9-1	>30>30
实施例13-1Example 13-1	>30>30
实施例14-1Example 14-1	>30>30

7.8 Experimental conclusion:

Inhibition of cardiac hERG potassium channel by drugs is the main reason for drug-induced QT prolongation syndrome. It can be seen from the experimental results that the compounds of the examples of the present invention have no obvious inhibitory effect on the cardiac hERG potassium ion channel, and can avoid the cardiotoxicity and side effects at high doses.

Test Example 8. Plasma Stability Test Protocol

8.1 Experimental purpose

The purpose of this experiment was to examine the stability of the compounds of the examples in mouse, rat, canine and human plasma.

8.2 Experimental procedure

8.2.1 Solution Preparation

1), plasma preparation

After animal or human whole blood was collected, it was put into a test tube containing anticoagulant, centrifuged at 3500 rpm for 10 min, and the upper pale yellow plasma was collected.

2), 10 μM test compound (m/M/V=C)

Compounds were weighed, stock solutions were made up in DMSO, and working solutions were made up in 100 mM phosphate buffer.

3), 10μM positive control

(1) Propantheline (Mr=449.4Da)

Weigh 2.36 mg of brommethamine and dilute it with 1 mL of DMSO to a 10 mM stock solution; pipette 10 μL of 10 mM stock solution into 1 mL of 100 mM phosphate buffer, the final concentration is 100 μM.

(2) Mevinolin (Lovastatin Mr=404.5Da)

Weigh 4.05 mg of lovastatin and dilute with 1 mL of DMSO to make a 10 mM stock solution; pipette 10 μL of 10 mM stock solution into 1 mL of 100 mM phosphate buffer to a final concentration of 100 μM.

8.2.2 Experimental process:

1) In a 96-well plate, add 285 μL of plasma and 15 μL of 10 μM compound (test compound) in sequence, and incubate at 37°C.

2) At 0, 15, 30, 60, 90, and 120 min (points can be fine-tuned), take out 40 μL respectively, and add 160 μL of acetonitrile stop solution containing internal standard.

3) After centrifugation (3500rpm, 10min), take 50μL of supernatant, add 50μL of DDH2O to dilute, and inject by LC-MS/MS.

8.3 Chromatographic conditions

Instrument: Shimadzu LC-20AD

Column: Phenomenex

C18 (50*4.6mm, 5μm particle size);

Mobile phase: A: acetonitrile, B: 0.1% formic acid solution 0～8min: 5%A→95%A, 2.0～2.1min: 90%A→5%A; flow rate: 0.8mL/min; running time: 5.0min ; Injection volume: 5 μL.

8.4 Mass Spec Conditions:

Instrument: API4000 liquid chromatography mass spectrometer, American AB company;

The ion source is electrospray ionization (ESI);

Drying gas (N2) temperature 500℃;

Electrospray voltage is 5500V;

The detection method is positive ion detection;

The scanning mode is selected reaction monitoring (MRM) mode;

The scan time is 0.1s.

8.5 Experimental results:

Table 16: Example Compound Plasma Stability Results

8.6 Experimental conclusion:

The above data show that the compounds of the examples of the present invention have high plasma stability and little difference between species.

Test Example 9, CYP enzyme single point inhibition test

9.1 Experimental purpose

Using the human liver microsome incubation system, the single-point method was used to rapidly predict the inhibition of CYP450 enzyme isoforms by compounds.

9.2 Experimental procedure

9.2.1 Solution Preparation

2.5mM NADPH, weigh 4.165mg NADPH (reduced nicotinamide adenine dinucleotide phosphate) and add 100mM phosphate buffer to 2mL. 0.25mg/mL microsomes, 50μL 20mg/mL microsomes, add 4mL 100mM phosphate buffer, mix well.

Preparation of test compound reaction solution

The compounds of the examples to be tested were weighed, diluted to 10 mM with DMSO, and then diluted to 100 μM with 100 mM phosphate buffer.

9.2.2 Experimental process:

1. In a 96-well plate, add 40 μL of liver microsomes, 10 μL of substrate, and 10 μL of the compound to be tested, and pre-incubate for 3 min.

2. Add 40 μL of NADPH.

3. Add 300 μL of acetonitrile stop solution containing internal standard at 20 min.

4. Centrifugal injection.

9.3 Experimental results:

Table 17: Results of single-point inhibition of CYP enzymes by example compounds

Note:

Strong inhibition: _IC50 <1 μM; Moderate inhibition: 1 μM < _IC50 <10 μM; Weak inhibition: _IC50 >10 μM

9.4 Experimental conclusion:

The above data show that the compounds of the examples of the present invention do not strongly inhibit each CYP enzyme subtype, and the risk of DDI is small.

Test Example 10. Plasma Protein Binding Rate Experiment

10.1 Experimental purpose:

The purpose of this experimental method is to detect the plasma protein binding of the compounds of the examples in plasma.

10.2 Experimental instruments and materials:

LC-MS, centrifuge, vortexer, pipette, continuous pipette, 96-well plate, tissue homogenizer (used for tissue sample analysis), 50% methanol aqueous solution, and acetonitrile solution of internal standard , blank matrix (plasma, urine or tissue homogenate, etc.)

10.3 Experimental steps:

10.3.1 Preparation of analyte stock solution A

Example compounds were formulated as 1 mM solutions in DMSO A.

10.3.2 Preparation of plasma solutions B

Take solution A and add it to the plasma solution to prepare 5uM solution B.

10.3.3 Processing flow

1) Add 200uL of solution B into the membrane.

2) Add 350uL PBS outside the membrane.

3) Incubate in a 37°C water bath for 6h.

4) The sample is processed and diluted and subjected to mass spectrometry detection.

10.4 Chromatographic conditions:

Instrument: Shimadzu LC-20AD;

Column: Phenomenex

C18 (50*4.6mm, 5μm particle size);

Mobile phase: A: acetonitrile, B: 0.1% formic acid solution 0～0.5min: 5%A→90%A, 2.0～2.1min: 90%A→5%A; flow rate: 0.8mL/min; running time: 5.0 min; injection volume: 5 μL.

10.5 Mass Spec Conditions:

The ion source is electrospray ionization (ESI);

Drying gas (N2) temperature 500℃;

Electrospray voltage is 5500V;

The detection method is positive ion detection;

The scanning mode was selected reaction monitoring (MRM) mode; the scanning time was 0.1s.

10.6 Experimental results:

Table 18: Example Compounds Plasma Protein Binding

编号Numbering	人people	大鼠rat	小鼠mouse	犬dog
实施例2-1Example 2-1	98.098.0	90.590.5	88.488.4	82.682.6

实施例9-1Example 9-1	99.899.8	94.994.9	90.190.1	98.798.7
实施例13-1Example 13-1	99.799.7	97.997.9	93.993.9	98.798.7
实施例14-1Example 14-1	96.896.8	95.495.4	96.396.3	92.592.5

10.7 Experimental conclusion:

The above data show that the compounds of the examples of the present invention show high plasma protein binding rate, and the species difference is small.

Test Example 11. Determination of Pharmacokinetics in Tumor-bearing Mice

11.1. Research purposes:

MiaPaca 2 tumor-bearing mice were used as test animals to study the pharmacokinetics of compound Example 13-1 and AMG-510 compound, orally administered in mice (plasma, tumor tissue and intestinal tract) at a dose of 6 mg/kg Dynamic behavior.

11.2. Test protocol

11.2.1 Test drug:

Example 13-1 of the present invention, AMG-510 compound, self-made.

11.2.2 Test animals:

MiaPaca 2 tumor-bearing mice 24, female. 3 mice at each time point (0h, 1h, 2h, 4h, 6h, 8h, 16h, 24h). Shanghai Sipple-Bikai Laboratory Animal Co., Ltd., animal production license number (SCXK (Shanghai) 2018-0006.

11.2.3 Drug preparation:

Weigh 5g of hydroxymethyl cellulose, dissolve it in 1000mL of purified water, and add 10g of Tween80. Mix well into a clear solution.

Example compound 13-1 was weighed, AMG-510 was dissolved in the solution, shaken, and sonicated for 15 minutes to obtain a uniform suspension with a concentration of 0.6 mg/mL.

11.2.4 Administration:

MiaPaca 2 tumor-bearing mice were fasted according to their body weight p.o. (the animals were not administered at 0 h), the dose was 6 mg/kg, and the administration volume was 10 mL/kg.

11.2.5 Sample collection:

Before and after administration, the mice were sacrificed by _CO2 , 0.5 ml of blood was collected from the heart, placed in an EDTA-2K test tube, centrifuged at 6000 rpm at 4 °C for 6 min to separate the plasma, and stored at -80 °C; the tumor tissue was weighed and placed in 2 mL in a centrifuge tube and stored at -80°C. The duodenum, ileum, and colon tissue were cut in appropriate lengths with scissors, the contents were removed, washed twice with PBS, dried with absorbent paper, weighed, placed in a 2 mL centrifuge tube, and stored at -80°C.

11.3 Experimental results: The LCMS/MS method was used to obtain the final determination results, as shown in Table 11:

Table 19: Mouse Pharmacokinetic Parameters of Compounds of the Invention

11.4 Experimental conclusion:

At the dose of 6 mg/kg, the ratio of the compound of the present invention in tumor exposure to blood exposure was higher than that of AMG-510, and the T _1/2 and MRT were longer.

3. Study on the salts of the compounds and their crystal forms

It is well known to those of ordinary skill in the art that when the compounds of the above examples are proved to have a good proliferation inhibitory effect on NCI-H358 and Mia PaCa-2 cells, their pharmaceutically acceptable salts often have the same pharmacological effects active. On this basis, the inventors further studied the physicochemical properties of the salt forms and crystal forms of the corresponding compounds, but the preparation and characterization of the following specific salt forms or crystal forms do not represent a limitation on the scope of the protection scope of the present invention, and those of ordinary skill in the art Based on the present invention, more salt forms and crystals of the compounds of the present invention can be obtained by conventional salt-forming or crystallization means, and these salt forms and crystals are all schemes protected by the present invention. details as follows:

1. Experimental equipment

1.1 Some parameters of physical and chemical testing instruments

1.2 Instrument and liquid analysis conditions

1.2.1 Instruments and Equipment

仪器名称equipment name	型号model
分析天平Analytical Balances	METTLER TOLEDO XA105METTLER TOLEDO XA105
纯水机water purifier	Milli-Q Plus，MilliporeMilli-Q Plus, Millipore
高效液相色谱仪High performance liquid chromatography	Thermo Ultimate 3000Thermo Ultimate 3000

1.2.2 Chromatographic conditions

2. Research on the salt form of the compound

2.1 Screening of the salt form of the compound of Example 13-1

2.1.1 Experimental purpose:

Screen compounds for salt forms.

2.1.2 Experimental steps:

1) Instruments and equipment

名称name	型号model	来源source
分析天平Analytical Balances	XA105XA105	METTLER TOLEDOMETTLER TOLEDO
超声波清洗仪Ultrasonic cleaner	SK5200LHCSK5200LHC	上海科导超声仪器Shanghai Kedao Ultrasound Instrument

pipette

Eppendorf (50mL, 100μL)

Eppendorf

2) Operation procedure

① Dissolve or suspend into salt

Weigh 10 mg of the compound, add 200 μL of solvent, stir at room temperature, add different acids respectively, stir overnight, centrifuge to dry or evaporate to dryness to obtain the salt of the compound.

②Anti-solvent method for salt formation

Select the good solvent, weigh the acid, add the good solvent to prepare a stock solution containing a compound concentration of 100 mg/mL, add the anti-solvent, weigh 100 mg of the compound separately, add 1 mL of the good solvent, dissolve all of them, and filter, and take 0.2 mL of the filtrate. , respectively drop the anti-solvent (stop adding if there is precipitation, add 1.8 mL of anti-solvent at most), stir for a period of time, remove the filtrate by rapid centrifugation, and obtain the salt of the compound.

2.1.3 Experimental results:

Through the salt type screening experiment, the compounds that can form salts with the free base of the compounds are sulfuric acid, isethionic acid, and 1,5-naphthalenedisulfonic acid.

As mentioned above, those skilled in the art can obtain more pharmaceutically acceptable salts by using conventional methods on the basis of the present invention.

2.2 Quantitative analysis of compound isethionate in Example 13-1

2.2.1 Quantification of isethionate by HPLC

2.2.1.1 Experimental purpose:

The number of isethionate in the compound isethionate of Example 13-1 was determined.

2.2.1.2 Experimental steps:

1) Chromatographic conditions

2) Operation

Weigh an appropriate amount of the free base of Example 13-1, add methanol to prepare a series of linear solutions with a concentration in the range of 0.05-0.30 mg/mL.

An appropriate amount of the isethionate compound of Example 13-1 was weighed, and methanol was added to prepare a solution containing the isethionate of Example 13-1 with a concentration of 0.25 mg/mL. Take the above-mentioned linear solution and sample solution for injection respectively.

2.2.1.3 Experimental results:

The calculation results of external standard method show that isethionic acid and free base form a salt in a molar ratio of 1:1.

2.2.2 Example 13-1 Compound isethionate ELSD quantification

2.2.2.1 Experimental purpose:

2.2.2.2 Experimental steps:

1) Chromatographic conditions

DilutentDiludent	MeOHMeOH
ColumnColumn	ZIC-HILIC(1504.6mm,5μm)ZIC-HILIC(1504.6mm,5μm)
Mobile phaseMobile phase	75mM醋酸铵溶液(pH4.80)：乙腈＝30：7075mM ammonium acetate solution (pH 4.80):acetonitrile=30:70
Injection volumeInjection volume	5μL5μL
Flow rateFlow rate	1.0mL/min1.0mL/min
Column TemperatureColumn Temperature	35℃35℃

ELSD TemperatureELSD Temperature	40℃40℃

2) Operation

Weigh an appropriate amount of isethionic acid, add methanol to prepare a series of linear solutions containing isethionic acid in the range of 0.5-1 mg/mL.

An appropriate amount of the compound isethionate compound of Example 13-1 was weighed, and methanol was added to prepare a solution containing the compound isethionate of Example 13-1 with a concentration of 5.0 mg/mL. Take the above-mentioned linear solution and sample solution for injection respectively.

2.2.2.3 Experimental results:

The number of isethionates in the isethionate of the compound of Example 13-1 is calculated to be 1.

3. Study on the crystal form of compound salt

3.1 Study on the crystal form of the compound of Example 13-1

3.1.1 Experimental purpose:

The compounds are screened for crystalline salts.

3.1.2 Experimental steps:

1) Instruments and equipment

2) Operation procedure

① Dissolve or suspend in different solvents to form salt crystals

Weigh 10 mg of the compound of Example 13-1, add different reaction solvents respectively, the final total volume is 200 μL, stir, add acid, stir for 12 hours, centrifuge and dry, and measure its XRPD.

② anti-solvent method salt crystallization

Select the good solvent, weigh the acid, add the good solvent to prepare a stock solution containing a compound concentration of 100 mg/mL, add the anti-solvent, weigh 100 mg of the compound separately, add 1 mL of the good solvent, dissolve all of them, and filter, and take 0.2 mL of the filtrate. , respectively drop the anti-solvent (stop adding if there is precipitation, add up to 1.8 mL of anti-solvent), stir for a period of time, quickly centrifuge to remove the filtrate, and measure the XRPD after the solid is dried.

3.1.3 Experimental results

Through the crystal form research experiment of the compound salt, the obtained salt forms with crystal form are isethionate, sulfate and 1,5-naphthalene disulfonate.

3.2 Preparation of the crystal form of the compound of Example 13-1

3.2.1 Experimental purpose:

The crystalline form of the compound salt of Example 13-1 was prepared.

3.2.2 Experimental steps:

1) Instruments and equipment

2) Operation procedure

I, the preparation of isethionate crystal form I

Weigh 500 mg of the compound of Example 13-1, add 9.08 mL of isopropanol, heat and stir at 50°C, add 0.914 mL of isethionic acid (1.0 M in MeOH), and precipitate out after dissolving, stir at room temperature for 2 hours, and filter The solid was dried under vacuum at 50°C to obtain isethionate crystal form I, which has the XRPD pattern shown in Figure 1, the DSC pattern shown in Figure 2 and the TGA shown in Figure 3 after detection and analysis picture.

Alternatively, add the compound of Example 13-1 (100g) and isopropanol (1200mL) into a 3L three-necked flask, heat to 40-45°C, stir to dissolve; then add 2-hydroxyethylsulfonic acid (28.84g) Disperse in 800 mL of ethanol, drop into the reaction system, control the temperature to 39-42 °C, and finish adding in about 10 minutes. 500 mg of seed crystals were added to the above reaction solution, and a solid was rapidly precipitated. The heating was removed, and the mixture was cooled to 25° C. and stirred for 12 h. Filter, wash the filter cake with 400 mL of isopropanol, dry it, and place it at 45°C for vacuum drying for 16 h to obtain 92.57 g of a pale yellow solid with a purity of 97.9%, a chiral purity of 99.8%, and a mass yield: 92%. After detection and analysis, it has an XRPD diagram basically as shown in FIG. 1 , a DSC diagram basically as shown in FIG. 2 , and a TGA diagram basically as shown in FIG. 3 .

II. Preparation of Isethionate Form II

Weigh 10 mg of the compound of Example 13-1, add 0.2 mL of tetrahydrofuran, heat and stir at 50 °C, add 18.3 μL of isethionic acid (1.0 M in MeOH), and precipitate out after dissolving, and stir at room temperature for 2 hours. Vacuum drying at 50° C. to obtain isethionate crystal form II, which has the XRPD pattern shown in FIG. 4 , the DSC pattern shown in FIG. 5 and the TGA pattern shown in FIG. 6 after detection and analysis.

III, the preparation of isethionate crystal form III

Weigh 20 mg of isethionate crystal form I, add 0.2 mL of methanol, 0.45 mL of methyl tert-butyl ether, heat and stir at 50°C overnight, and filter the solid under vacuum drying at 50°C to obtain isethionate. Form III, after detection and analysis, has the XRPD pattern shown in FIG. 7 , the DSC pattern shown in FIG. 8 and the TGA pattern shown in FIG. 9 .

IV, the preparation of sulfate crystal form I

Weigh 10 mg of the compound of Example 13-1, add 0.2 mL of ethanol, heat and stir at 50 °C, add 18.3 μL of sulfuric acid (1.0 M in MeOH), dissolve and clear out a precipitate, stir at room temperature overnight, and filter the solid under vacuum drying at 50 °C. , to obtain sulfate crystal form I, which has the XRPD pattern shown in Figure 10 after detection and analysis.

V, the preparation of sulfate crystal form II

Weigh 100 mg of the compound of Example 13-1, add 1.82 mL of isopropanol, heat and stir at 50 °C, add 183 μL of sulfuric acid (1.0 M in MeOH), dissolve and clear out a solid, stir at room temperature overnight, and filter the solid at 50 °C under vacuum conditions After drying, the sulfate crystal form II is obtained, which has the XRPD pattern shown in FIG. 11 after detection and analysis.

VI, the preparation of sulfate crystal form III

Weigh 10 mg of sulfate crystal form I, add 0.2 mL of isopropanol, heat and stir at 50°C for 5 days, and filter the solid under vacuum drying at 50°C to obtain sulfate crystal form III. The XRPD graph shown.

VII, the preparation of sulfate crystal form IV

Weigh 10 mg of sulfate crystal form I, add 0.2 mL of ethyl acetate, heat and stir at 50 °C for 5 days, and filter the solid under vacuum drying at 50 °C to obtain sulfate crystal form III, which has the characteristics shown in Figure 13 after detection and analysis. The XRPD graph shown.

4. Solid Stability Experiment

4.1 The immobilization stability experiment of the compound isethionate crystal form I of Example 13-1

4.1.1 Experimental purpose:

The physical and chemical stability of the crystal form of the compound under the conditions of high temperature, high humidity, high temperature and high humidity and light was investigated to provide the basis for the screening and storage of the crystal form.

4.1.2 Instrument and liquid analysis conditions

4.1.3 Experimental scheme

4.1.3.1 Weigh an appropriate amount of the isethionate crystal form I of the compound of Example 13-1, respectively, under light (≥1.2×106lux·h, 10 days), high humidity (25°C, 75%, 10 days), The XRPD was measured after being treated for a certain period of time under conditions of high humidity (25°C, 90%, 10 days), high temperature (40°C, 30 days), high temperature (60°C, 30 days) and micropowder.

4.1.4.1 Experimental results:

4.1.3.2 Experimental scheme:

Weigh an appropriate amount of the isethionate crystal form I of the compound of Example 13-1, respectively, under illumination (5000±500lux), high temperature (60°C), high humidity (92.5% RH), high temperature and high humidity (50°C & 75% After standing for 10 days under RH) conditions, add diluent methanol to prepare a solution containing the free base of Example 13-1 with a concentration of 0.25 mg/mL, analyze by HPLC, and calculate the changes of related substances according to the peak area normalization method.

4.1.4.2 Experimental results:

The above experimental results show that the isethionate crystal form I of the compound of Example 13-1 is relatively stable under the conditions of light, high humidity, high temperature and micropowder.

4.2 Solid Stability Experiment of Compound Sulfate Form II of Example 13-1

4.2.1 Experimental purpose:

4.2.2 Instrument and liquid analysis conditions

4.2.3 Experimental scheme:

Weigh an appropriate amount of the sulfate crystal form II of the compound of Example 13-1, respectively, under the conditions of light (5000±500lux), high temperature (60°C), high humidity (92.5%RH), high temperature and high humidity (50°C&75%RH) After standing for 10 days, add diluent methanol to prepare a solution containing the free base of Example 13-1 with a concentration of 0.25 mg/mL, analyze by HPLC, and calculate the changes of related substances according to the peak area normalization method.

4.2.4 Experimental results:

The sulfate crystal form II is relatively stable under the conditions of light, high humidity, high temperature and high humidity.

5. Solubility experiments in different media

5.1 The solubility test of the compound of Example 13-1 in different media

5.1.1 Experimental purpose:

Investigation of isethionate crystal form I and sulfate crystal form II in different pH media, water, artificial simulated gastric fluid (FaSSGF), fasting artificial simulated intestinal fluid (FaSSIF) and non-fasting artificial simulated intestinal fluid (FeSSIF), etc. The solubility in the medium provides a basis for the evaluation of the druggability of the salt.

5.1.2 Experimental scheme:

About 1 mg of different salt forms of the compound were weighed and suspended in 1 mL of artificial simulated gastric fluid (FaSSGF), fasting artificial simulated intestinal fluid (FaSSIF), non-fasting artificial simulated intestinal fluid (FeSSIF) and pure water for 24 hours, and detected by HPLC. The external standard method was used to determine the thermodynamic solubility of the compounds at 37°C.

5.1.3 Experimental results: as shown in the following table:

6. Thermodynamic stability experiment

6.1 Polymorphic screening of the isethionate salt of the compound of Example 13-1

6.1.1 Experimental purpose:

Through polymorphic screening, a thermodynamically stable crystal form of isethionate was obtained.

6.1.2 Experimental scheme:

Take 10 mg of isethionate crystal form I, add 200 μL of organic solvent respectively, beating for 5 days at room temperature and 50 ° C, centrifuge, discard the supernatant, and measure the XRPD after the solid is dried.

6.1.3 Experimental results: the following table:

The above results show that isethionate crystal form I is a stable crystal form of isethionate.

6.2 The polymorphic screening experiment of the sulfate salt of the compound of Example 13-1

6.2.1 Experimental purpose:

Through polymorphic screening, a thermodynamically stable sulfate crystal form was obtained.

6.2.2 Experimental scheme:

Take 10 mg of sulfate crystal form II, add 200 μL of organic solvent respectively, beat at 50 °C for 5 days, centrifuge, discard the supernatant, and measure the XRPD after the solid is dried.

6.2.3 Experimental results: the following table:

序号serial number	溶剂solvent	硫酸盐Sulfate
--	初始晶型initial crystal form	晶型IIForm II
11	乙醇Ethanol	晶型IIForm II
22	2-甲基四氢呋喃2-Methyltetrahydrofuran	晶型IIForm II
33	2-丁酮2-Butanone	晶型IIForm II
44	乙酸乙酯Ethyl acetate	晶型IIForm II
55	甲苯Toluene	晶型IIForm II
66	乙酸异丙酯isopropyl acetate	晶型IIForm II
77	叔丁醇tert-Butanol	晶型IIForm II

The above results show that sulfate crystal form II is a stable crystal form of sulfate.

Claims

The acid salt of the compound represented by the general formula (II),

in:

Ra is selected from hydrogen or methyl;

R 1 is selected from hydrogen, fluorine, chlorine, bromine or methyl;

R 3 is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, methyl, -S(CH 3 ) or trifluoromethyl;

R 4 is selected from hydrogen, amino, hydroxyl, fluorine, chlorine, -N(CH 3 ) 2 , -NH(CH 3 ) or fluorine;

R 5 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl;

R 6 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl or isopropyl;

R 7 is selected from hydrogen, fluorine, chlorine, bromine or methyl;

The acid of the acid salt is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetylaminobenzoic acid, 4-aminobenzoic acid, capric acid, hexanoic acid acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid acid, pyroglutamic acid, tartaric acid, lauryl sulfuric acid, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid , glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethyl acetate Alkanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid.
The acid salt of the compound according to claim 1, wherein the compound is further represented by the general formula (II-A) or (II-B):
The acid salt of the compound according to claim 1 or 2, wherein the compound is selected from:
The acid salt of the compound according to any one of claims 1-3, wherein the compound is

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6 -Chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-iso propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-iso propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1 -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1 -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

The acid in the acid salt is selected from isethionic acid, sulfuric acid, 1,5-naphthalenedisulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleic acid, adipic acid, hydrochloric acid , citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid or fumaric acid, preferably isethionic acid or sulfuric acid.
The acid salt of the compound according to any one of claims 1-4, wherein the number of acids is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1 , 2 or 3, more preferably 1.
The acid salt of the compound according to any one of claims 1-5, wherein the acid salt is a hydrate or an anhydrate; when the acid salt is a hydrate, the number of water is 0.2-3; Preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
The acid salt of the compound according to any one of claims 1-6, wherein the acid salt is a crystal form;

Preferred compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl) -Acid salt crystal form of 6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one ;

P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-iso Acid salt form of propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-iso Acid salt form of propyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1 -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one acid salt crystalline form;

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1 -(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one acid salt crystalline form;

More preferably isethionate crystal form, sulfate crystal form, 1,5-naphthalene disulfonate crystal form, mesylate crystal form, hydrobromide salt crystal form, phosphate crystal form, benzenesulfonic acid Salt crystal form, oxalate crystal form, maleate crystal form, adipate crystal form, hydrochloride crystal form, citrate crystal form, malonate crystal form, L-malate crystal form , Palmoate crystal form, p-toluenesulfonate crystal form or fumarate crystal form.
The acid salt of compound according to claim 7, is characterized in that:

P-4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6 The crystal form of the acid salt of -chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is:

Isethionate crystal form I, its X-ray powder diffraction pattern 2θ has a diffraction peak at 21.7±0.2°; or has a diffraction peak at 8.8±0.2°; or has a diffraction peak at 19.3±0.2°; or has a diffraction peak at 27.6±0.2°; or has a diffraction peak at 10.9±0.2°; or has a diffraction peak at 15.4±0.2°; or has a diffraction peak at 16.7±0.2°; or has a diffraction peak at 15.8±0.2° or a diffraction peak at 17.5±0.2°; or a diffraction peak at 23.8±0.2°; or a diffraction peak at 10.2±0.2°; or a diffraction peak at 11.8±0.2°; preferably Include any 2-5 places, or 3-5 places, or 3-6 places, or 3-8 places, or 5-8 places, or 6-8 places in the above-mentioned diffraction peaks, more preferably include any 6 places, 7 or 8;

It is isethionate crystal form II, and its X-ray powder diffraction pattern 2θ has a diffraction peak at 21.7±0.2°; or a diffraction peak at 8.8±0.2°; or a diffraction peak at 19.3±0.2° or a diffraction peak at 27.6±0.2°; or a diffraction peak at 10.9±0.2°; or a diffraction peak at 23.8±0.2°; or a diffraction peak at 16.7±0.2°; or a diffraction peak at 15.4±0.2° There is a diffraction peak at 0.2°; or a diffraction peak at 15.8±0.2°; or a diffraction peak at 10.0±0.2°; preferably any 2-5, or 3-5, or 3 of the above-mentioned diffraction peaks are included -6 places, or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

It is isethionate crystal form III, and its X-ray powder diffraction pattern 2θ has a diffraction peak at 19.4±0.2°; or a diffraction peak at 16.9±0.2°; or a diffraction peak at 26.6±0.2° or a diffraction peak at 14.6±0.2°; or a diffraction peak at 28.0±0.2°; or a diffraction peak at 25.6±0.2°; or a diffraction peak at 20.7±0.2°; or a diffraction peak at 12.8±0.2° There is a diffraction peak at 0.2°; or a diffraction peak at 19.1±0.2°; or a diffraction peak at 27.2±0.2°; preferably any 2-5, or 3-5, or 3 of the above-mentioned diffraction peaks are included -6 places, or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

It is sulfate crystalline form I, and its X-ray powder diffraction pattern 2θ has a diffraction peak at 19.0±0.2°; or has a diffraction peak at 19.4±0.2°; or has a diffraction peak at 12.4±0.2°; or at 12.4±0.2° or at 17.6±0.2°; or at 18.1±0.2°; or at 25.3±0.2°; or at 8.8±0.2° or have a diffraction peak at 21.9±0.2°; or have a diffraction peak at 11.5±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

It is sulfate crystal form II, and its X-ray powder diffraction pattern 2θ has a diffraction peak at 15.5±0.2°; or a diffraction peak at 11.1±0.2°; or a diffraction peak at 8.9±0.2°; or at 8.9±0.2° or at 22.3±0.2°; or at 23.6±0.2°; or at 17.4±0.2°; or at 27.3±0.2° or have a diffraction peak at 17.0±0.2°; or have a diffraction peak at 27.9±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

It is sulfate crystal form III, and its X-ray powder diffraction pattern 2θ has a diffraction peak at 19.6±0.2°; or has a diffraction peak at 18.0±0.2°; or has a diffraction peak at 18.4±0.2°; or at 18.4±0.2° or at 14.3±0.2°; or at 11.8±0.2°; or at 14.9±0.2°; or at 25.7±0.2° Diffraction peaks; or have diffraction peaks at 15.4±0.2°; or have diffraction peaks at 23.5±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8 places, or 5-8 places, or 6-8 places, more preferably including any 6 places, 7 places or 8 places;

It is sulfate crystal form IV, and its X-ray powder diffraction pattern has a diffraction peak at 19.4±0.2°; or has a diffraction peak at 18.9±0.2°; or has a diffraction peak at 15.5±0.2°; or has a diffraction peak at 8.8 or at 18.1±0.2°; or at 24.9±0.2°; or at 17.4±0.2°; or at 12.3±0.2° or have a diffraction peak at 26.1±0.2°; or have a diffraction peak at 14.5±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them.
The acid salt of compound according to claim 8, is characterized in that:

The X-ray powder diffraction pattern of isethionate crystal form I at least contains one or more diffraction peaks located at 2θ of 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, preferably two of them , more preferably three strips; optionally, it can further include 2θ at 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 10.2±0.2°, 11.8±0.2° At least one of °, preferably including 2, 3, 4 or 5 of them; for example,

8.8±0.2°, 27.6±0.2°;

21.7±0.2°, 8.8±0.2°, 10.9±0.2°;

21.7±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°;

15.8±0.2°, 8.8±0.2°, 27.6±0.2°, 10.9±0.2°; 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 15.8±0.2°, 10.9±0.2°, 15.4±0.2°;

10.9±0.2°, 8.8±0.2°, 10.2±0.2°, 27.6±0.2°, 10.9±0.2°, 15.8±0.2°;

The X-ray powder diffraction pattern of isethionate crystal form II contains at least one or more diffraction peaks located at 2θ of 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, preferably two of them , more preferably including three; optionally, it may further include at least one located at 2θ of 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, preferably including 2 bar, 3, 4 or 5; for example,

21.7±0.2°, 10.0±0.2°;

21.7±0.2°, 10.0±0.2°, 19.3±0.2°;

21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°;

21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 16.7±0.2°, 27.6±0.2°, 10.9±0.2°;

The X-ray powder diffraction pattern of isethionate crystal form III contains at least one or more diffraction peaks located at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, preferably two of them , more preferably including three; optionally, it can further include at least one located at 2θ of 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, preferably including 2 bar, 3, 4 or 5; for example,

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form I at least comprises one or more diffraction peaks located at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, preferably two of them, more preferably comprising Three; optionally, it may further comprise at least one of 2θ of 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, preferably two or three of them , 4 or 5; for example,

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form II contains at least one or more diffraction peaks located at 2θ of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, preferably two of them, more preferably Three strips; optionally, it can further comprise at least one located at 2θ of 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, preferably two or three of them , 4 or 5; for example,

15.5±0.2°, 11.1±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 27.3±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form III contains at least one or more diffraction peaks located at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, preferably two of them, more preferably three; optionally, it can further comprise at least one of 2θ of 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, preferably two or three of them , 4 or 5; for example,

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form IV contains at least one or more diffraction peaks located at 2θ of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, preferably two of them, more preferably Three; optionally, it may further comprise at least one of 2θ of 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, preferably two or three of them , 4 or 5; for example,

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°.
The acid salt of the compound according to claim 8 or 9, characterized in that:

The X-ray powder diffraction pattern of isethionate Form I optionally further comprises positions at 2θ of 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 19.3±0.2° One or more diffraction peaks in 0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 17.5±0.2°, 23.8±0.2°; preferably at least contain Wherein any 2-3, or 4-5, or 6-8; further preferably, including any 2, 3, 4, 5, 6, 7 or 8; for example,

8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.8±0.2°, 17.5±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 17.5±0.2°, 16.7±0.2°, 15.8±0.2°;

The X-ray powder diffraction pattern of isethionate Form II optionally further comprises positions at 2θ of 10.0±0.2°, 21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9+ One or more diffraction peaks in ±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 10.0±0.2°; preferably at least any 2-3 of them, or 4 -5 places, or 6-8 places; further preferably, including any 2 places, 3 places, 4 places, 5 places, 6 places, 7 places or 8 places; for example,

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9+±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°;

The X-ray powder diffraction pattern of isethionate Form III optionally further comprises positions at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2° One or more diffraction peaks in 0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°; preferably at least any of 2-3, or 4-5, or 6 -8 places; further preferably, including any 2, 3, 4, 5, 6, 7 or 8 places; for example,

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 27.2±0.2°;

The X-ray powder diffraction pattern of sulfate Form I optionally further comprises positions at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3 One or more diffraction peaks in ±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; Further preferably, it includes any 2, 3, 4, 5, 6, 7 or 8 of them; for example,

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°;

The X-ray powder diffraction pattern of the sulfate crystal form II optionally further comprises positions at 2θ of 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4 One or more diffraction peaks in ±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; Further preferably, it includes any 2, 3, 4, 5, 6, 7 or 8 of them; for example,

15.5±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form III optionally further comprises positions at 2θ of 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9 One or more diffraction peaks in ±0.2°, 25.7±0.2°, 15.4±0.2°, 23.5±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; Further preferably, it includes any 2, 3, 4, 5, 6, 7 or 8 of them; for example,

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 23.5±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form IV optionally further comprises positions at 2θ of 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4 One or more diffraction peaks in ±0.2°, 12.3±0.2°, 26.1±0.2°, 14.5±0.2°; preferably at least any 2-3, or 4-5, or 6-8 of them; Further preferably, it includes any 2, 3, 4, 5, 6, 7 or 8 of them; for example,

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°.
The acid salt of compound according to claim 8, is characterized in that:

The X-ray powder diffraction pattern of isethionate crystalline form I comprises positions at 2θ of 21.7±0.2°, 8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.1±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 13.3±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 17.5±0.2°, 23.8±0.2°, 14.7±0.2°, 24.3±0.2°, One or more diffraction peaks in 27.3±0.2°, 23.4±0.2°, 20.6±0.2°, 21.2±0.2°, preferably, including optional 4, 5, 6, 8 or 10 There are diffraction peaks at; for example,

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 20.6±0.2°;

8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 20.6±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 20.6±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 15.4±0.2°, 16.7±0.2°, 15.8±0.2°, 24.3±0.2°, 23.8±0.2°;

8.8±0.2°, 10.2±0.2°, 11.8±0.2°, 13.1±0.2°, 27.6±0.2°, 10.9±0.2°, 13.3±0.2°, 21.2±0.2°, 15.8±0.2°, 17.5±0.2°;

The X-ray powder diffraction pattern of isethionate crystalline form II comprises positions at 2θ of 21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, One or more of 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 17.5±0.2°, 14.7±0.2°, 24.4±0.2°, 27.3±0.2°, 29.2±0.2° Diffraction peaks, preferably, including optional 4, 5, 6, 8 or 10 diffraction peaks; for example,

10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 29.2±0.2°;

21.7±0.2°, 10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 29.2±0.2°;

21.7±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 27.3±0.2°, 17.5±0.2°;

10.0±0.2°, 8.8±0.2°, 19.3±0.2°, 27.6±0.2°, 10.9±0.2°, 23.8±0.2°, 16.7±0.2°, 15.4±0.2°, 15.8±0.2°, 17.5±0.2°;

The X-ray powder diffraction pattern of isethionate crystalline form III comprises positions at 2θ of 19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, One or more of 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°, 24.4±0.2°, 15.3±0.2°, 26.2±0.2°, 30.2±0.2°, 27.4±0.2° Diffraction peaks, preferably, including optional 4, 5, 6, 8 or 10 diffraction peaks; for example,

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 27.4±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 27.4±0.2°;

19.4±0.2°, 16.9±0.2°, 26.6±0.2°, 14.6±0.2°, 28.0±0.2°, 25.6±0.2°, 20.7±0.2°, 12.8±0.2°, 19.1±0.2°, 27.2±0.2°;

The X-ray powder diffraction pattern of sulfate crystalline form I contains positions at 2θ of 19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2° One or more diffraction peaks in , 8.8±0.2°, 21.9±0.2°, 11.5±0.2°, preferably, including optional 4, 5, 6, 8 or 10 diffraction peaks ;E.g,

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 11.5±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°;

19.0±0.2°, 19.4±0.2°, 12.4±0.2°, 26.2±0.2°, 17.6±0.2°, 18.1±0.2°, 25.3±0.2°, 8.8±0.2°, 21.9±0.2°, 11.5±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form II contains 2θ at 15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2° , 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 15.8±0.2°, 24.2±0.2°, 21.8±0.2°, 10.3±0.2°, 20.6±0.2° one or more diffraction peaks, Preferably, there are diffraction peaks at optional 4, 5, 6, 8 or 10 positions; for example,

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 21.8±0.2°;

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 20.6±0.2°, 27.9±0.2°;

15.5±0.2°, 11.1±0.2°, 8.9±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 20.6±0.2°, 27.9±0.2°;

11.1±0.2°, 8.9±0.2°, 19.3±0.2°, 22.3±0.2°, 23.6±0.2°, 17.4±0.2°, 27.3±0.2°, 17.0±0.2°, 27.9±0.2°, 20.6±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form III contains 2θ at 19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2° , 25.7±0.2°, 15.4±0.2°, 23.5±0.2°, 18.8±0.2°, 24.7±0.2°, 9.5±0.2°, 8.8±0.2°, 11.1±0.2° one or more diffraction peaks, Preferably, there are diffraction peaks at optional 4, 5, 6, 8 or 10 positions; for example,

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.1±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 11.1±0.2°;

19.6±0.2°, 18.0±0.2°, 18.4±0.2°, 16.8±0.2°, 14.3±0.2°, 11.8±0.2°, 14.9±0.2°, 25.7±0.2°, 15.4±0.2°, 23.5±0.2°;

The X-ray powder diffraction pattern of sulfate crystal form IV contains 2θ at 19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2° , 12.3±0.2°, 26.1±0.2°, 14.5±0.2°, 22.2±0.2°, 24.3±0.2°, 21.7±0.2°, 23.6±0.2° one or more diffraction peaks, preferably, including wherein Diffraction peaks at optional 4, 5, 6, 8, or 10; for example,

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 23.6±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 23.6±0.2°;

19.4±0.2°, 18.9±0.2°, 15.5±0.2°, 8.8±0.2°, 18.1±0.2°, 24.9±0.2°, 17.4±0.2°, 12.3±0.2°, 26.1±0.2°, 14.5±0.2°.
The acid salt of the compound according to any one of claims 8-11, wherein:

The X-ray powder diffraction pattern of isethionate Form I is shown in Figure 1;

The X-ray powder diffraction pattern of isethionate crystal form II is shown in Figure 4;

The X-ray powder diffraction pattern of isethionate crystal form III is shown in Figure 7;

The X-ray powder diffraction pattern of sulfate crystal form I is shown in Figure 10;

The X-ray powder diffraction pattern of sulfate crystal form II is shown in Figure 11;

The X-ray powder diffraction pattern of sulfate crystal form III is shown in Figure 12;

The X-ray powder diffraction pattern of sulfate crystal form IV is shown in FIG. 13 .
The acid salt of the compound according to any one of claims 8-11, wherein:

In the X-ray powder diffraction pattern of isethionate crystal form I, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 1 are ±0.2°～±0.5°, preferably ±0.2 °～±0.3°, most preferably ±0.2°;

In the X-ray powder diffraction pattern of isethionate crystal form II, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 4 are ±0.2°～±0.5°, preferably ±0.2 °～±0.3°, most preferably ±0.2°;

In the X-ray powder diffraction pattern of isethionate crystal form III, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 7 are ±0.2°～±0.5°, preferably ±0.2 °～±0.3°, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form I, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 10 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form II, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 11 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form III, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 12 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°;

In the X-ray powder diffraction pattern of sulfate crystal form IV, the relative peak intensity of the top ten diffraction peak positions and the 2θ error of the diffraction peak at the corresponding position in Figure 13 are ±0.2°～±0.5°, preferably ±0.2°～±0.3 °, most preferably ±0.2°.
The acid salt of the compound according to any one of claims 1-13, wherein the isethionate crystal form I has the DSC spectrum shown in Figure 2;

Isethionate crystal form II has the DSC spectrum shown in Figure 5;

Isethionate Form III has the DSC pattern shown in FIG. 8 .
The acid salt of the compound according to any one of claims 1-14, wherein the crystal form of the acid salt is a hydrate or an anhydrate, and when the crystal form of the acid salt is a hydrate, the number of water is 0.2-3, preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3; further, the water in the hydrate is pipeline water or crystal water or a combination of both.
The method for preparing the acid salt of the compound described in any one of claims 1-15, comprising the steps:

1) Weigh an appropriate amount of free base, add reaction solvent to dissolve;

2) Add an appropriate amount of acid and stir; the amount of acid is preferably 1.2 equivalents;

3) after centrifugal drying, obtain compound acid salt or its crystal form;

The reaction solvent is an organic solvent, preferably ethanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, At least one of ethyl acetate or 1,4-dioxane;

Acid selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethanesulfonic acid, dichloro Acetic acid, trichloroacetic acid, acetylhydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetylaminobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, mandelic acid, pyrovalley Amino acid, tartaric acid, dodecyl sulfate, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid , 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethanesulfonic acid, Benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid.
The method for preparing the acid salt of the compound described in any one of claims 1-15, comprising the steps:

1) Weigh an appropriate amount of free base, add reaction solvent to dissolve;

2) Add an appropriate amount of acid and organic solvent and stir to dissolve;

3) optionally, adding seed crystals;

4) cooling, filtering out the solid, washing with solvent, and drying;

The reaction solvent used in step 1) is an organic solvent, preferably ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, At least one of n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;

The acid in step 2) is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, ethane Sulfonic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetylaminobenzoic acid, 4-aminobenzoic acid, capric acid, Caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, erythorbic acid, lactic acid, malic acid, Mandelic acid, pyroglutamic acid, tartaric acid, dodecyl sulfate, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentian acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methyl alcohol Sulfonic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-amino water succinic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably hydrochloric acid, phosphoric acid, ethanesulfonic acid, benzenesulfonic acid, methanesulfonic acid, fumaric acid, isethionic acid, oxalic acid or hydrobromic acid;

The organic solvent in step 2) is selected from one or more of alcohols, ethers, ketones or ester solvents, preferably ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl alcohol at least one of tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;

The solvent in step 3) is selected from one or more of alcohols, ethers, ketones or ester solvents, preferably ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl alcohol At least one of tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane.
A pharmaceutical composition comprising a therapeutically effective amount of an acid salt of a compound of any one of claims 1-15, and one or more pharmaceutically acceptable carriers, diluents or excipients.
Use of the acid salt of the compound according to any one of claims 1-15 or the pharmaceutical composition of claim 18 in the preparation of a KRAS inhibitor drug; preferably in the preparation of a RAS G12C mutation inhibitor drug .
The acid salt of the compound according to any one of claims 1-17 or the pharmaceutical composition according to claim 18 is used for the treatment of Noonan syndrome, Leopard skin syndrome, leukemia, neuroblastoma, melanoma, Use in medicines for diseases or conditions such as esophageal cancer, head and neck cancer, breast cancer, lung cancer and colon cancer; preferably non-small cell lung cancer, colon cancer, esophageal cancer and head and neck cancer.